Antibacterial properties of experimentally produced birch tar and its medicinal affordances in the Pleistocene

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Drawing from recent arguments suggesting multimodal uses of products such as ochre and birch tar, this study tests the antibiotic properties of birch tar produced experimentally with methods reconstructed for Middle Palaeolithic birch tar finds from Europe. Made from the bark of Betula pendula and Betula pubescens , widely documented for the European Late Pleistocene, we produced birch tar samples using an underground pit method, a condensation method, and a modern tin can method. The birch tar samples were tested for antibiotic properties using the modified Kirby-Bauer disc diffusion antibiotic assay. The results indicate a moderate effect against the Gram-positive Staphylococcus aureus , a bacterium widely known for its role in wound infections. We further establish that the efficacy of antibiotic properties is not related to the production method, with all methods showing a degree of variation. This supports a coevolutionary relationship between medicinal and technological use and production of birch tar during the Pleistocene. The antibiotic properties documented in this study are consistent with the use of birch tar as a wound dressing and skin ointment in Mi'kmaq communities in Eastern Canada, and the use of birch tar in Saami communities of Lapland. Arguing from an underexplored angle between experimental archaeology and ethnopharmacology, we suggest that similar to the ethnographic examples, a use of birch tar beyond exclusively technological contexts must be considered for the Middle Palaeolithic. Early Pyrotechnology Birch Tar Ethnopharmacology Neanderthal Medicine Transformative Technologies Experimental Archaeology Figures Figure 1 Figure 2 Figure 3 Figure 4 1 Introduction Birch tar is a well-known component of Neanderthal life, and its documented use in the Late Pleistocene primarily includes its use as a hafting agent ( 1 ). As such, traces of it have been found on archaeological sites in Germany, along the Dutch coast, and in Italy ( 2 – 7 ). Produced under thermolytic and anaerobic conditions in fired clay pits, the complex production process was likely preceded by generations of experimentation and material engagement leading up to production methods with a higher yield ( 3 ). In other regions of the world, conifer resin, beeswax, bitumen, Podocarpus tar, and ochre have been found to have been used as hafting agents in Middle Palaeolithic or Middle Stone Age contexts ( 8 – 15 ). Ochre has not only been discussed as a component of hafting agents but is also discussed to have been used in various other contexts, such as body paint, but also regarding its UV protective and insect repellent properties ( 16 , 17 ). Birch tar, conversely, has received little attention beyond its use in hafting contexts within Pleistocene contexts ( 18 , 19 ). This may partly be due to terminological inconsistencies: Whilst being referred to as birch tar in contexts relating to Pleistocene adhesives, it is also discussed as birch extract elsewhere and carries a number of Indigenous names ( 20 ). Its use as an adhesive and wound dressing is known, for example, from Yakut and Saami communities ( 21 , 22 ). In Mi’kmaq communities Indigenous to Eastern Canada, it is known as maskwio’mi – translated as birch bark oil – and used for its medicinal properties. Here, the production of maskwio’mi is a practice known through oral history passed down from Elders, and the production method involving tins has recently been reconstructed ( 23 ). The practice likely would have involved ceramic production similar to that known from the European Neolithic until a few generations ago ( 24 , 25 ). Previous studies have indicated broad-spectrum antibiotic properties of maskwio’mi ( 26 , 27 ). Recent years have seen a surge of interest in Neanderthal life beyond stone tools, and today, the use of medicinal plants by Neanderthals is known from numerous contexts ( 28 – 32 ). At El Sidrón, Spain, isotope signatures corresponding to chamomile ( Matricaria chamomilla ) and yarrow ( Achillea millefolium ) were detected in the dental calculus of a Neanderthal individual and have been suggested to have been used for its medicinal properties ( 33 ). Yarrow was also detected at Shanidar Cave, Iraqi-Kurdistan ( 32 ). Shanidar also shows that communities of care date way beyond the Holocene, with a severed but healed tibia of a Neanderthal individual dependent on the care of the wider group ( 30 , 34 ). In this study, we analyse the antibacterial properties of birch tar produced experimentally using European birch variants and relying on methods reconstructed for use in Middle Palaeolithic contexts against Staphylococcus aureus (Gram-positive) and Escherichia coli (Gram-negative) using a modified Kirby-Bauer disc diffusion assay and discuss the implications of our results against current perspectives on structures of care in Neanderthal communities. 2 Materials and methods This section describes several extraction methods for producing birch tar through experimental processes with European birch varieties, namely Betula pendula and Betula pubescens , employing techniques reconstructed for Middle Palaeolithic settings. Using a modified Kirby-Bauer disc diffusion method, the antibacterial activity of the birch tar was evaluated against Staphylococcus aureus and Escherichia coli , Gram-positive and Gram-negative bacteria, respectively. 2.1 Birch tar production and extraction In producing the birch tar, we adhered to the reconstructions summarized by Schmidt et al. ( 3 , 35 ) and Bierenstiel et al. ( 23 ). Birch bark was obtained from both Betula pubescens and Betula pendula , the two species that would have been common during the European Pleistocene. The bark was collected by T. Siemssen on several occasions in different areas of Germany, including Lkrs. Lüchow-Dannenberg, Lkrs. Cuxhaven, and Rhein-Sieg-Kreis (see Table 1 ). The raw bark was obtained from dead trees on public land, and no permission was required for collection. Samples of both B. pubescens and B. pendula have been formally identified and deposited in the Herbarium of the University of Bonn (herbarium code BONN), and can be accessed via the accession numbers 4155 ( B. pendula ) and 4156 ( B. pubescens ). The bark was transformed into tar using three methods: Distillation in a tin, distillation in a raised clay structure, and the condensation method (Fig. 1). Figure 1. Different methods for Neanderthal birch tar production have been reconstructed by Schmidt et al. ( 3 ). 1: Birch bark. 2: Birch tar. a: Birch tar piece found at the Middle Palaeolithic site of Königsaue, Germany. b: Cobble grove condensation method. c: Buried bark roll method. d: Condensation method. e: Pit roll method. f: Raised structure method. Methods b and f have been used in this study. Modified after: Schmidt et al. ( 3 ). Destructive distillation of birch tar in a tin involved adding densely packed birch bark to a tin with a lid, and piercing holes into the bottom of the tin. A second, smaller container was placed underneath the larger tin to collect the tar formed during the distillation process. A fire was lit on top of the tin, heating the tin for approximately 120 minutes (Fig. 2). A tinfoil spoon was used to extract the birch tar from the bottom container. This method is well-documented in historical contexts and produces reliable and consistent results ( 26 , 27 ). Distillation of birch tar in a raised structure involved placing a small container in the bottom of a small hole, then adding densely packed birch tar (c. 150 g dried bark has been used for the setups of this study) on top of it and encapsulating it with a thin layer of clay. A fire was then lit on the structure, burning for approximately 120 minutes. The structure was then taken off, and the birch tar was extracted from the bottom container using a tinfoil spoon to minimise contamination. During the production of birch tar using the raised structure method, any contamination by secondary plant products was avoided by using tin foil instead of foliage for the construction of pits to obtain the cleanest samples possible. A commercially available clay (Claytec® Clay topcoat fine 06) was chosen for the construction of the pits to ensure homogenous fine fibre content across all experiments. For a detailed description of the setup of the raised structure method, see Kozowyk et al. ( 36 ). The condensation method involves burning smaller amounts of birch bark under a fireproof surface, such as stone. The condensed birch tar was subsequently collected on the surface and scraped off using a folded tinfoil spoon to minimize contamination of the sample. This method yields smaller amounts of birch tar compared to the two other methods but has been theorised to have been used in the initial stages of birch tar production in Neanderthal contexts ( 3 , 35 ). Figure 2. Distillation of birch bark in a tin in Cape Breton (Unama’ki), Nova Scotia. This method is known from oral accounts of Mi’kmaq Elders and was used to produce maskwio’mi, an ointment used to treat wounds and skin conditions. Photograph by Nicolaas Honig. 2.2 Antimicrobial assay We obtained S. aureus ATCC 25923 and E. coli ATCC 25922 strains from the American Type Culture Collection (ATCC®; USA). Using these two strains allowed to compare efficacy against both a Gram-positive and a Gram-negative strain, since earlier analysis of birch tar (maskwio’mi) produced in a controlled laboratory environment showed broad-spectrum antibiotic properties against both (see section 4.1 ). The strains were inoculated onto TSA (Sigma-Aldrich) plates at 37°C for 24 h. The obtained pure cultures were suspended in 0.85% sterile normal saline. The bacterial suspensions were adjusted to achieve a 0.5 McFarland standard. All work about the handling of bacterial cultures was performed in a Labconco® Purifier Microbiological Safety Cabinet Class II type A2. The antibacterial activities of the birch tar were determined by using the modified Kirby-Bauer disc diffusion antibiotic assay ( 37 , 38 ). We saturated overnight a blank, sterile Whatman Filter paper disc (6 mm in diameter) with 20 µL of birch tar with a concentration ranging from 30–400 mg/mL in DMSO. Suitable bacterial suspensions were uniformly distributed on Mueller-Hinton agar (MHA, Sigma-Aldrich) plates using a sterile disposable cotton swab. Discs containing bark extracts were placed on the inoculated agar plates with sterile tweezers. The standard antibiotics- Gentamicin (10 µg / disc), BBL, 6 mm Sensi-disc- were used as the positive control, and DMSO as the negative control. The entire antibacterial assay was carried out under strict aseptic conditions. Experiments were performed in duplicate. The plates were incubated overnight at 37°C. A clear zone of inhibition (ZOI) was measured in millimetres with a ruler and reported as average measurements according to Clinical and Laboratory Standards Institute (CLSI) guidelines ( 39 ). Detailed figures of ZOI experiments (Figures S1 to S6) can be found in the supplementary material section. 3 Results The birch tar (BT) yield of the six samples, BT1 - BT6, varied significantly across samples, ranging from 0.06 g to 0.69 g (Table 1 ). We tested the antibacterial potential of birch tar using a modified version of the Kirby-Bauer disc diffusion method. The results (see Table 2 ) showed that birch tar was selectively active and worked against Staphylococcus aureus but had no effect on Escherichia coli . None of the samples produced any measurable inhibition zones for E. coli (0 ± 0.0 mm), indicating no antibacterial activity against this Gram-negative bacterium. In contrast, the birch tar samples showed varying inhibition levels against S. aureus . The effectiveness depended on both the extraction method and the concentration used. Sample BT5 stood out, showing the highest antibacterial activity with a zone of inhibition measuring 10.5 ± 0.7 mm. This sample was made using the raised structure method with Betula pendula bark at a concentration of 133 mg/mL. Other samples (BT1, BT2, BT4, and BT6) showed moderate activity, with inhibition zones between 7.0 and 7.5 mm. One sample, BT3, showed no activity, mirroring its lack of effect on E. coli . As expected, the standard antibiotic Gentamicin (10 µg/disc) used as a positive control was more effective, producing clear zones of 22–23 mm against both bacteria. The negative control (DMSO) showed no inhibition. Figure 3 . Photos of sample ID BT_2024_012 − 01 (BT5) showing antibacterial activity against Staphylococcus aureus and Escherichia coli. a: Zone of inhibition of BT5 showing antibacterial activity against Gram-positive Staphylococcus aureus ATCC 25923. b: Zone of inhibition of BT5 with no antibacterial activity against Gram-negative Escherichia coli ATCC 25922. Table 1 Details of samples obtained from birch tar extraction using different methods and bark species. OrigID Extraction method Bark species Sample date obtained Source Latitude Source Longitude Sample Yield (g) BT_2024_001–01 Tin Can Modern Betula pubescens 2/12/2023 53.54 8.75 0.39 BT_2024_005 − 01 Condensation Betula pendula 2/27/2024 50.83 7.13 0.11 BT_2024_006 − 01 Condensation Betula pubescens 2/27/2024 53.54 8.75 0.06 BT_2024_011 − 01 Raised Structure Betula pendula 10/3/2024 50.83 7.13 0.33 BT_2024_012 − 01 Raised Structure Betula pendula 10/3/2024 52.94 11.26 0.69 BT_2024_013 − 01 Raised Structure Betula pubescens 10/3/2024 53.54 8.75 0.59 Table 2. Zone of inhibition (ZOI, mm) of birch tar (BT1-6) against Staphylococcus aureus and Escherichia coli. 4 Discussion Here, we discuss our samples in direct comparison with samples obtained by other studies to establish differences related to the production method and birch species and consider the implications of our results for Middle Palaeolithic healthcare practices. 4.1 Comparative analysis Our results show that birch tar has a selective antibacterial effect, working specifically against Gram-positive bacteria like Staphylococcus aureus . The inhibition zones we recorded ranged from 7.0 ± 0.0 mm to 10.5 ± 0.7 mm, suggesting a moderate but clear activity (see Table 2 ). This pattern aligns with evidence from other plant-based antimicrobials, which generally exhibit more potent effects against Gram-positive bacteria. The primary explanation for this is their structural variation: Gram-positive bacteria show a thicker peptidoglycan layer, whereas Gram-negative bacteria have an additional outer membrane that can prevent antimicrobial entry ( 40 ). Our findings not only reinforce what past scientific studies have reported, but they also echo traditional knowledge. Birch tar has long been recognised for its antiseptic properties, particularly in treating skin infections, and our study adds new evidence to support its potential use in modern and historical healthcare practices. Similarly, the outcomes align with the traditional practices of the L’nu (Mi’kmaq) people, who have used birch bark extract to address skin infections commonly caused by S. aureus ( 41 – 45 ). Earlier research has shown that birch tar contains a range of phenolic derivatives, such as catechols and guaiacols ( 27 , 44 , 45 ), which are likely responsible for its antibacterial and antifungal effect ( 46 , 47 ). Indeed, Richert et al. ( 48 ) reported that phenolic compounds in birch tar contribute substantially to its antimicrobial activity, with pronounced effects on Gram-positive bacteria. The selective inhibition of S. aureus found in our research thus aligns well with the known antimicrobial capabilities of phenols ( 46 , 49 , 50 ). This relationship has also been investigated by Agarwal et al. ( 51 ), who assessed how polyphenolic content relates to antibacterial effects by comparing bark extracts of wild cherry, larch, and sweet chestnut against S. aureus and E. coli . Their bacterial growth curves indicated that the extracts did not suppress E. coli ; in fact, bacterial proliferation was even enhanced. In contrast, S. aureus was strongly inhibited. Their sweet chestnut extract was most effective which they linked to its high antioxidant potential and highest total phenol content compared to wild cherry and European larch. None of the samples showed measurable inhibition of E. coli (0 ± 0 mm), implying limited efficacy against Gram-negative species, likely due to lower phenolic levels and the protective barriers of their outer membrane. This selectivity may also be a result of the distinctions in cell wall structures between Gram-positive and Gram-negative bacteria, reflecting variations in cell surface ( 52 ) and the outer membrane structure (53). Hitherto unpublished data by Kaliaperumal et al. ( 26 ), who have assessed the antibacterial properties of industrially produced maskwio’mi made from Betula papyrifera , compares well to the results presented here. The antibacterial activity of their extracts TE1 and TE2 revealed higher inhibition zones against S. aureus strains (18–20 mm for TE1 and 11–12 mm for TE2) compared to our BT samples, which ranged from 7.0 to 10.5 mm. The higher inhibition zones of their extracts may suggest that TE1 and TE2, particularly TE1, possess more potent bioactive compounds than our BT samples. Kaliaperumal et al. ( 26 ) identified over 60 bioactive compounds, including phenols, monoterpenes, and triterpenes, in the chemical analyses (GC-MS) results of their extracts. Based on the GC-MS analysis, the authors attributed the higher bioactivity of TE1 to its elevated triterpene concentration, including betulin and lupeol derivatives, which are known for their antimicrobial effects ( 51 ). In contrast, their extract TE2 exhibited lower activity, potentially due to its higher naphthalene derivative content, which may be less effective against Gram-positive bacteria. This may explain the highest antibacterial activity against S. aureus recorded for our sample BT5 (10.5 ± 0.7 mm ZOI) in comparison with other samples BT1, BT2, BT4, and BT6 (7.0 ± 0.0 to 7.5 -± 0.7 mm) in our study, which may suggest that BT5 contains higher concentrations of bioactive compounds than others. Phenolic compounds appear to be key contributors to the antimicrobial activity of birch tar, although volatile compounds may also have an effect ( 54 ). Further analysis of GC-MS on the birch tar samples would be ideal to validate the compounds present in our samples. Just like our samples, the samples TE1 and TE2 extracts displayed weak activity against Gram-negative bacteria, with minimal inhibition against E. coli , including Klebsiella pneumoniae , and Pseudomonas aeruginosa . This further supports the hypothesis that the antimicrobial compounds primarily target Gram-positive bacterial species. Several previous studies have examined the antibacterial properties of related extracts. Acquaviva et al. ( 55 ) reported that the extract of Betula aetnensis leaves inhibited S. aureus but had a lower impact on Gram‑negative bacteria. Similarly, Emrich et al. ( 56 ) reported that water-based extracts of Betula pendula were highly effective against S. epidermidis and MRSA. Vandal et al. ( 57 ), too, showed that ethanol extracts from plant material of Betula papyrifera inhibited S. aureus but not E. coli . 4.2 Implications for Neanderthal healthcare The differences among the six samples collected for our study indicate varying extraction efficiencies, which could be due to factors like the composition of the raw materials, the exact conditions during pyrolysis, or natural variability in the properties of birch bark. Importantly, there is no clear relationship between the extraction method and antibacterial efficacy. Samples produced using the condensation method and the raised structure method result in similar ZOI diameters against gram-positive Staphylococcus aureus widely known for its role in wound infections. Consequently, the application of birch tar to the skin specifically for the treatment of wounds and skin conditions would have been afforded as early as exploration of its hafting properties has occurred, minimally during MIS 7 from c. 191–243 ka [5,57], and does not rely on underground pit production. Given the low viscosity of birch tar produced in underground pits, and adhesive properties of birch tar, contamination of the skin during handling is nearly inevitable, regardless of production technique. Indeed, quantities of birch tar sufficient for skin application are low, with 0.2 g of birch tar covering as much as 100 cm 2 of skin (19; see Fig. 4), and are thus easily obtained as a by-product of production for its use in a hafting context, regardless of the production process. In this regard, it might prove useful to consider further uses along with skin application, as previously discussed for ochre ( 16 , 17 ). For example, studies have suggested the efficacy of birch tar as an insect-repellent [58; Faraone et al., unpublished data], and the Late Pleistocene environments of Europe saw considerable ecological and epidemiological pressure. This is evidenced by, for example, the extensive flying insect assemblages known from Neanderthal sites such as Lichtenberg, or Salzgitter-Lebenstedt ( 19 , 60 , 61 ) as well as by genetic material relating to immune response introgressed from Neanderthals ( 62 – 65 ). Figure 4. A thin layer of viscous birch tar distributed on a white surface. Approximately 0.2 g of birch tar are sufficient to cover c. 100 cm 2 of skin surface. This study on birch tar’s affordances for wound care sits in the context of a surge in interest in Neanderthal life beyond stone tools. Structures of care are increasingly recognized as an essential part of Pleistocene life, and numerous scholars have now published on neglected aspects of Neanderthal care. Spikins et al. ( 29 , 30 ) have explored structures of care in the Neanderthal context. Houldcraft and Underdown ( 66 ) have documented the plethora of pathogens that Neanderthals objected to during the Middle Palaeolithic. Hardy ( 31 ) and Hardy et al. ( 33 ) have documented the manifold plants used potentially medicinally found in Neanderthal contexts. Weyrich et al. ( 67 ) have documented dental care in a Neanderthal individual from El Sidròn. Trinkaus and Villotte ( 68 ) and Conde-Valverde et al. ( 69 ) have reported on structures of care related to disability in Neanderthal communities. Today, practice of care is understood to be a key component of Neanderthal life. As such, our study contributes more explicit data on the multimodal affordances of pyrotechnological birch tar production, shedding light on the healthcare of deep time. 5 Conclusion Our experimental findings demonstrate that birch tar possesses selective antibacterial properties, showing consistent inhibitory effects against Staphylococcus aureus but no detectable activity against Escherichia coli. Among the six birch tar samples tested, only BT5—extracted via the raised structure method from Betula pendula —showed a comparatively strong response, producing an inhibition zone of 10.5 ± 0.7 mm. Other samples exhibited mild to moderate activity, while one (BT3) showed no effect. This variability underscores how differences in extraction method, bark species, and possibly compound concentration influence the antibacterial efficacy of birch tar. The complete absence of inhibition against E. coli across all samples aligns with the known structural resistance of Gram-negative bacteria. Further, it supports the tar’s specificity toward Gram-positive strains. These results reinforce the hypothesis that birch tar’s antibacterial properties may have been exploited intentionally in both deep time and Indigenous contexts for wound care, and bear potential for targeted therapeutic development in the present day. Nonetheless – our study permits exploring the co-evolutionary relationship between technology and medicine as early as MIS 7, since all three tested production methods showed some level of antibacterial properties, and offers more explicit data to support the medicinal use of antibacterial birch tar in deep time. Whilst drawing from arguments previously proposed for the use of ochre in the Pleistocene, such multimodal uses may indeed be considered for other aspects and localities of Pleistocene lifeways. As such, the use of, for example, biomass adhesive made from Podocarpus during the Middle Stone Age of southern Africa may also be considered in light of the well-documented antibacterial properties of Podocarpus plant extract ( 12 , 70 – 72 ). Yet, further research going beyond the exploratory character of this study is necessary, that further isolates the parameters that affect the antibiotic activity of birch tar produced using different methods. As today’s world is facing an antibiotic crisis that sees increased antibiotic tolerance of bacterial strains ( 73 , 74 ), engagement with traditional remedies becomes ever more important. The detection of novel antibacterial remedies (or those that have been neglected in Western medicine) is seen as a key aspect of reconciling with the increasing antibiotic resistance. There is global demand for both broad-spectrum antibiotics that cover both Gram-positive and Gram-negative bacteria strains, as well as more targeted antibacterial remedies that are strain-specific to avoid the administration of broad-spectrum antibiotics, such as the one documented in this study. Along with the affordances of care in deep time explored by our study, this may further lead to the development of therapies specific to S. aureus . Declarations Author Contribution Statement: TS: Conceptualisation, Formal analysis, Investigation, Methodology, Writing - original draft. AO: Data curation, Formal analysis, Investigation, Methodology, Writing - original draft. MS: Investigation, Writing - review & editing. JP: Investigation, Writing - review & editing. MB: Conceptualisation, Funding acquisition, Methodology, Project administration, Supervision, Writing - review & editing. Funding Tjaark Siemssen is funded by the Boise Trust of the University of Oxford and the German Academic Scholarship Foundation ( Studienstiftung ). Aderonke Oludare was funded by Canadian Institutes of Health Research (CIHR) Project Grant 420598 awarded to Matthias Bierenstiel. Data Availability Statement All relevant data are within the manuscript and its Supporting Information files. Samples of Betula pubescens and Betula pendula used in this study can be accessed in the herbarium of the University of Bonn, Germany (herbarium code BONN) via the accession numbers 4155 ( B. pendula ) and 4156 ( B. pubescens ). References Roebroeks W, Soressi M. Neandertals revised. Proc Natl Acad Sci USA. 2016;113(23):6372–9. Pawlik AF, Thissen JP. Hafted armatures and multi-component tool design at the Micoquian site of Inden-Altdorf, Germany. Journal of Archaeological Science. 2011;38(7):1699–708. Schmidt P, Koch TJ, Blessing MA, Karakostis FA, Harvati K, Dresely V, et al. 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Memorial University of Newfoundland; 2025 Blondeau D, St‐Pierre A, Bourdeau N, Bley J, Lajeunesse A, Desgagné‐Penix I. Antimicrobial activity and chemical composition of white birch (Betula papyrifera Marshall) bark extracts. MicrobiologyOpen. 2020;9(1):e00944. Richert A, Olewnik-Kruszkowska E, Dąbrowska GB, Dąbrowski HP. The role of birch tar in changing the physicochemical and biocidal properties of polylactide-based films. IJMS. 2021;23(1):268. Richert A, Olewnik-Kruszkowska E, Malinowski R, Kalwasińska A, Swiontek Brzezinska M. Polycaprolactone-based films incorporated with birch tar—thermal, physicochemical, antibacterial, and biodegradable properties. Foods. 2023;12(23):4244. Barbieri R, Coppo E, Marchese A, Daglia M, Sobarzo-Sánchez E, Nabavi SF, et al. Phytochemicals for human disease: An update on plant-derived compounds antibacterial activity. Microbiological Research. 2017;196:44–68. Hasler Gunnarsdottir S, Sommerauer L, Schnabel T, Oostingh GJ, Schuster A. Antioxidative and antimicrobial evaluation of bark Extracts from common European Trees in light of dermal applications. Antibiotics. 2023;12(1):130. Agarwal C, Hofmann T, Vršanská M, Schlosserová N, Visi-Rajczi E, Voběrková S, et al. In vitro antioxidant and antibacterial activities with polyphenolic profiling of wild cherry, the European larch and sweet chestnut tree bark. Eur Food Res Technol. 2021;247(9):2355–70. Cetin-Karaca H, Newman MC. Antimicrobial efficacy of plant phenolic compounds against Salmonella and Escherichia Coli. Food Bioscience. 2015;11:8–16. Olasupo NA, Fitzgerald DJ, Gasson MJ, Narbad A. Activity of natural antimicrobial compounds against Escherichia coli and Salmonella enterica serovar Typhimurium. Lett Appl Microbiol. 2003;37(6):448–51. Hagner M, Pasanen T, Lindqvist B. Effects of birch tar oils on soil organisms and plants. AFSci. 2008;19(1):13. Acquaviva R, Menichini F, Ragusa S, Genovese C, Amodeo A, Tundis R, et al. Antimicrobial and antioxidant properties of Betula aetnensis Rafin. (Betulaceae) leaves extract. Natural Product Research. 2013;27(4–5):475–9. Emrich S, Schuster A, Schnabel T, Oostingh GJ. Antimicrobial activity and wound-healing capacity of birch, beech and larch bark extracts. Molecules. 2022;27(9):2817. Vandal J, Abou-Zaid MM, Ferroni G, Leduc LG. Antimicrobial activity of natural products from the flora of Northern Ontario, Canada. Pharmaceutical Biology. 2015;53(6):800–6. Bonitto E, Smith KL, Bierenstiel M, Kaliaperumal R, Goralski K. Investigating birch bark oil anti-oxidative properties in human Keratinocytes. The Journal of Pharmacology and Experimental Therapeutics. 2023;385:355. Thorsell W, Mikiver A, Malander I, Tunón H. Efficacy of plant extracts and oils as mosquito repellents. Phytomedicine. 1998;5(4):311–23. Hein M, Urban B, Tanner DC, Buness AH, Tucci M, Hoelzmann P, et al. 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Zhou S, Butler-Laporte G, Nakanishi T, Morrison DR, Afilalo J, Afilalo M, et al. A Neanderthal OAS1 isoform protects individuals of European ancestry against COVID-19 susceptibility and severity. Nat Med. 2021;27(4):659–67. Houldcroft CJ, Underdown SJ. Neanderthal genomics suggests a Pleistocene time frame for the first epidemiologic transition. American J Phys Anthropol. 2016;160(3):379–88. Weyrich LS, Duchene S, Soubrier J, Arriola L, Llamas B, Breen J, et al. Neanderthal behaviour, diet, and disease inferred from ancient DNA in dental calculus. Nature. 2017;544(7650):357–61. Trinkaus E, Villotte S. External auditory exostoses and hearing loss in the Shanidar 1 Neandertal. Rosenberg K, editor. PLOS ONE. 2017;12(10):e0186684. Conde-Valverde M, Quirós-Sánchez A, Diez-Valero J, Mata-Castro N, García-Fernández A, Quam R, et al. The child who lived: Down syndrome among Neanderthals? Sci Adv. 2024;10(26):eadn9310. Abdillahi HS, Stafford GI, Finnie JF, Van Staden J. Antimicrobial activity of South African Podocarpus species. Journal of Ethnopharmacology. 2008;119(1):191–4. Abdillahi HS, Finnie JF, Van Staden J. Anti-inflammatory, antioxidant, anti-tyrosinase and phenolic contents of four Podocarpus species used in traditional medicine in South Africa. Journal of Ethnopharmacology. 2011;136(3):496–503. Cock IE, Van Vuuren SF. The traditional use of southern African medicinal plants for the treatment of bacterial respiratory diseases: A review of the ethnobotany and scientific evaluations. Journal of Ethnopharmacology. 2020;263:113204. Aggarwal R, Mahajan P, Pandiya S, Bajaj A, Verma SK, Yadav P, et al. Antibiotic resistance: a global crisis, problems and solutions. Critical Reviews in Microbiology. 2024;50(5):896–921. Church NA, McKillip JL. Antibiotic resistance crisis: challenges and imperatives. Biologia. 2021;76(5):1535–50. Additional Declarations The authors declare no competing interests. Supplementary Files Siemssenetal2025PLOSONESupplementaryInformation.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8153400","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":547420073,"identity":"6832c078-9b4f-490e-9ccc-8afb735da5e6","order_by":0,"name":"Tjaark Siemssen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6ElEQVRIie2RMQrCMBSGUwpxCWaNtOgVKgWhUOhVWoROqUfQSiFdBNceJyWgSw+QVWeHgotgBxN1lDi4OOQLPLJ8/O/nAWCx/CVOqSfCanD1wFh9iElAb8WflG8FfldexAEHz5zvSoLF9iyHNQpl0XLQxVPolY7sTSkkq8KCCbSQq5QDmYfQ527UGBWHeUXJlUIDMfQiYySFHjIpuK3vVC/WUFWnF5unMpgUkDGXQhcFRCtSpFArpvpIZpWnu5DuopQunzPVLtoZlNH+eLqqxRJc07AHh3iGybKVN1PMB17HtVgsFssPPACIuEs7CbtdmAAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0009-0003-0210-1040","institution":"Institute of Archaeology, University of Oxford, UK; Institute for Prehistory, University of Cologne, Germany","correspondingAuthor":true,"prefix":"","firstName":"Tjaark","middleName":"","lastName":"Siemssen","suffix":""},{"id":547420659,"identity":"984b181b-8767-4103-9d7f-0c636881acc5","order_by":1,"name":"Aderonke Oludare","email":"","orcid":"https://orcid.org/0000-0001-8657-7166","institution":"Department of Chemistry, Cape Breton University, Canada","correspondingAuthor":false,"prefix":"","firstName":"Aderonke","middleName":"","lastName":"Oludare","suffix":""},{"id":547420660,"identity":"569685ef-6ef4-49e4-be8d-98215a24f4b6","order_by":2,"name":"Marcel Schemmel","email":"","orcid":"https://orcid.org/0000-0002-0435-7651","institution":"TraceoLab, University of Liège, Belgium","correspondingAuthor":false,"prefix":"","firstName":"Marcel","middleName":"","lastName":"Schemmel","suffix":""},{"id":547420661,"identity":"3e9d53f9-b92c-43d8-8a39-6cd0dad59be5","order_by":3,"name":"Janos Puschmann","email":"","orcid":"https://orcid.org/0009-0003-6778-8488","institution":"Institute for Prehistory, University of Cologne, Germany; 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Methods b and f have been used in this study. Modified after: Schmidt et al. (3).\u003c/p\u003e","description":"","filename":"SiemssenOludareBierenstiel2025Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-8153400/v1/56244d46b18c7dae2e89132c.png"},{"id":97108448,"identity":"31b4ed97-4eb8-432d-a68f-6c1e02c288d7","added_by":"auto","created_at":"2025-12-01 05:42:43","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":7762657,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDistillation of birch bark in a tin in Cape Breton (Unama’ki), Nova Scotia.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis method is known from oral accounts of Mi’kmaq Elders and was used to produce maskwio’mi, an ointment used to treat wounds and skin conditions. Photograph by Nicolaas Honig.\u003c/p\u003e","description":"","filename":"SiemssenOludareBierenstiel2025Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-8153400/v1/b3ddda3a102c249a9cb9aed6.png"},{"id":97108446,"identity":"e4f35ed8-d71e-4293-b601-1dbf638dd648","added_by":"auto","created_at":"2025-12-01 05:42:43","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2294991,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhotos of sample ID BT_2024_012-01 (BT5) showing antibacterial activity against\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e Staphylococcus aureus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e Escherichia coli.\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003ea: Zone of inhibition of BT5 showing antibacterial activity against Gram-positive \u003cem\u003eStaphylococcus aureus\u003c/em\u003e ATCC 25923. b: Zone of inhibition of BT5 with no antibacterial activity against Gram-negative\u003cem\u003e Escherichia coli\u003c/em\u003e ATCC 25922.\u003c/p\u003e","description":"","filename":"SiemssenOludareBierenstiel2025Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-8153400/v1/b5a55a4754e04ccb9cd6dfdb.png"},{"id":97108450,"identity":"66347266-2e7f-4899-b821-e2ce1ec8e16b","added_by":"auto","created_at":"2025-12-01 05:42:43","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":4225061,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA thin layer of viscous birch tar distributed on a white surface.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eApproximately 0.2 g of birch tar are sufficient to cover c. 100 cm\u003csup\u003e2\u003c/sup\u003e of skin surface.\u003c/p\u003e","description":"","filename":"SiemssenOludareBierenstiel2025Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-8153400/v1/db75605168f87d17570ee528.png"},{"id":97249023,"identity":"09f4481d-0726-4c3a-8cec-3453868d197b","added_by":"auto","created_at":"2025-12-02 13:09:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":15608071,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8153400/v1/6ddb18d4-e908-4e08-80df-95f33aca9211.pdf"},{"id":97108447,"identity":"d942e7cc-1d7a-4506-ba53-6a36a5172739","added_by":"auto","created_at":"2025-12-01 05:42:43","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":666872,"visible":true,"origin":"","legend":"","description":"","filename":"Siemssenetal2025PLOSONESupplementaryInformation.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8153400/v1/a5d856955cd0fe89cbee63c8.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eAntibacterial properties of experimentally produced birch tar and its medicinal affordances in the Pleistocene\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eBirch tar is a well-known component of Neanderthal life, and its documented use in the Late Pleistocene primarily includes its use as a hafting agent (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). As such, traces of it have been found on archaeological sites in Germany, along the Dutch coast, and in Italy (\u003cspan additionalcitationids=\"CR3 CR4 CR5 CR6\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Produced under thermolytic and anaerobic conditions in fired clay pits, the complex production process was likely preceded by generations of experimentation and material engagement leading up to production methods with a higher yield (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn other regions of the world, conifer resin, beeswax, bitumen, Podocarpus tar, and ochre have been found to have been used as hafting agents in Middle Palaeolithic or Middle Stone Age contexts (\u003cspan additionalcitationids=\"CR9 CR10 CR11 CR12 CR13 CR14\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Ochre has not only been discussed as a component of hafting agents but is also discussed to have been used in various other contexts, such as body paint, but also regarding its UV protective and insect repellent properties (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eBirch tar, conversely, has received little attention beyond its use in hafting contexts within Pleistocene contexts (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). This may partly be due to terminological inconsistencies: Whilst being referred to as \u003cem\u003ebirch tar\u003c/em\u003e in contexts relating to Pleistocene adhesives, it is also discussed as \u003cem\u003ebirch extract\u003c/em\u003e elsewhere and carries a number of Indigenous names (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Its use as an adhesive and wound dressing is known, for example, from Yakut and Saami communities (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). In Mi\u0026rsquo;kmaq communities Indigenous to Eastern Canada, it is known as maskwio\u0026rsquo;mi \u0026ndash; translated as birch bark oil \u0026ndash; and used for its medicinal properties. Here, the production of maskwio\u0026rsquo;mi is a practice known through oral history passed down from Elders, and the production method involving tins has recently been reconstructed (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). The practice likely would have involved ceramic production similar to that known from the European Neolithic until a few generations ago (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). Previous studies have indicated broad-spectrum antibiotic properties of maskwio\u0026rsquo;mi (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eRecent years have seen a surge of interest in Neanderthal life beyond stone tools, and today, the use of medicinal plants by Neanderthals is known from numerous contexts (\u003cspan additionalcitationids=\"CR29 CR30 CR31\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). At El Sidr\u0026oacute;n, Spain, isotope signatures corresponding to chamomile (\u003cem\u003eMatricaria chamomilla\u003c/em\u003e) and yarrow (\u003cem\u003eAchillea millefolium\u003c/em\u003e) were detected in the dental calculus of a Neanderthal individual and have been suggested to have been used for its medicinal properties (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). Yarrow was also detected at Shanidar Cave, Iraqi-Kurdistan (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). Shanidar also shows that communities of care date way beyond the Holocene, with a severed but healed tibia of a Neanderthal individual dependent on the care of the wider group (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn this study, we analyse the antibacterial properties of birch tar produced experimentally using European birch variants and relying on methods reconstructed for use in Middle Palaeolithic contexts against \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (Gram-positive) and \u003cem\u003eEscherichia coli\u003c/em\u003e (Gram-negative) using a modified Kirby-Bauer disc diffusion assay and discuss the implications of our results against current perspectives on structures of care in Neanderthal communities.\u003c/p\u003e"},{"header":"2 Materials and methods","content":"\u003cp\u003eThis section describes several extraction methods for producing birch tar through experimental processes with European birch varieties, namely \u003cem\u003eBetula pendula\u003c/em\u003e and \u003cem\u003eBetula pubescens\u003c/em\u003e, employing techniques reconstructed for Middle Palaeolithic settings. Using a modified Kirby-Bauer disc diffusion method, the antibacterial activity of the birch tar was evaluated against \u003cem\u003eStaphylococcus aureus\u003c/em\u003e and \u003cem\u003eEscherichia coli\u003c/em\u003e, Gram-positive and Gram-negative bacteria, respectively.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Birch tar production and extraction\u003c/h2\u003e\u003cp\u003eIn producing the birch tar, we adhered to the reconstructions summarized by Schmidt et al. (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e) and Bierenstiel et al. (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). Birch bark was obtained from both \u003cem\u003eBetula pubescens\u003c/em\u003e and \u003cem\u003eBetula pendula\u003c/em\u003e, the two species that would have been common during the European Pleistocene. The bark was collected by T. Siemssen on several occasions in different areas of Germany, including Lkrs. L\u0026uuml;chow-Dannenberg, Lkrs. Cuxhaven, and Rhein-Sieg-Kreis (see Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The raw bark was obtained from dead trees on public land, and no permission was required for collection. Samples of both \u003cem\u003eB. pubescens\u003c/em\u003e and \u003cem\u003eB. pendula\u003c/em\u003e have been formally identified and deposited in the Herbarium of the University of Bonn (herbarium code BONN), and can be accessed via the accession numbers 4155 (\u003cem\u003eB. pendula\u003c/em\u003e) and 4156 (\u003cem\u003eB. pubescens\u003c/em\u003e). The bark was transformed into tar using three methods: Distillation in a tin, distillation in a raised clay structure, and the condensation method (Fig.\u0026nbsp;1).\u003c/p\u003e\u003cp\u003e\u003cb\u003eFigure\u0026nbsp;1. Different methods for Neanderthal birch tar production have been reconstructed by Schmidt et al.\u003c/b\u003e (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e1: Birch bark. 2: Birch tar. a: Birch tar piece found at the Middle Palaeolithic site of K\u0026ouml;nigsaue, Germany. b: Cobble grove condensation method. c: Buried bark roll method. d: Condensation method. e: Pit roll method. f: Raised structure method. Methods b and f have been used in this study. Modified after: Schmidt et al. (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eDestructive distillation of birch tar in a tin involved adding densely packed birch bark to a tin with a lid, and piercing holes into the bottom of the tin. A second, smaller container was placed underneath the larger tin to collect the tar formed during the distillation process. A fire was lit on top of the tin, heating the tin for approximately 120 minutes (Fig.\u0026nbsp;2). A tinfoil spoon was used to extract the birch tar from the bottom container. This method is well-documented in historical contexts and produces reliable and consistent results (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eDistillation of birch tar in a raised structure involved placing a small container in the bottom of a small hole, then adding densely packed birch tar (c. 150 g dried bark has been used for the setups of this study) on top of it and encapsulating it with a thin layer of clay. A fire was then lit on the structure, burning for approximately 120 minutes. The structure was then taken off, and the birch tar was extracted from the bottom container using a tinfoil spoon to minimise contamination. During the production of birch tar using the raised structure method, any contamination by secondary plant products was avoided by using tin foil instead of foliage for the construction of pits to obtain the cleanest samples possible. A commercially available clay (Claytec\u0026reg; Clay topcoat fine 06) was chosen for the construction of the pits to ensure homogenous fine fibre content across all experiments. For a detailed description of the setup of the raised structure method, see Kozowyk et al. (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe condensation method involves burning smaller amounts of birch bark under a fireproof surface, such as stone. The condensed birch tar was subsequently collected on the surface and scraped off using a folded tinfoil spoon to minimize contamination of the sample. This method yields smaller amounts of birch tar compared to the two other methods but has been theorised to have been used in the initial stages of birch tar production in Neanderthal contexts (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eFigure\u0026nbsp;2. Distillation of birch bark in a tin in Cape Breton (Unama\u0026rsquo;ki), Nova Scotia.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThis method is known from oral accounts of Mi\u0026rsquo;kmaq Elders and was used to produce maskwio\u0026rsquo;mi, an ointment used to treat wounds and skin conditions. Photograph by Nicolaas Honig.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Antimicrobial assay\u003c/h2\u003e\u003cp\u003eWe obtained \u003cem\u003eS. aureus\u003c/em\u003e ATCC 25923 and \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922 strains from the American Type Culture Collection (ATCC\u0026reg;; USA). Using these two strains allowed to compare efficacy against both a Gram-positive and a Gram-negative strain, since earlier analysis of birch tar (maskwio\u0026rsquo;mi) produced in a controlled laboratory environment showed broad-spectrum antibiotic properties against both (see section \u003cspan refid=\"Sec7\" class=\"InternalRef\"\u003e4.1\u003c/span\u003e). The strains were inoculated onto TSA (Sigma-Aldrich) plates at 37\u0026deg;C for 24 h. The obtained pure cultures were suspended in 0.85% sterile normal saline. The bacterial suspensions were adjusted to achieve a 0.5 McFarland standard. All work about the handling of bacterial cultures was performed in a Labconco\u0026reg; Purifier Microbiological Safety Cabinet Class II type A2. The antibacterial activities of the birch tar were determined by using the modified Kirby-Bauer disc diffusion antibiotic assay (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). We saturated overnight a blank, sterile Whatman Filter paper disc (6 mm in diameter) with 20 \u0026micro;L of birch tar with a concentration ranging from 30\u0026ndash;400 mg/mL in DMSO. Suitable bacterial suspensions were uniformly distributed on Mueller-Hinton agar (MHA, Sigma-Aldrich) plates using a sterile disposable cotton swab. Discs containing bark extracts were placed on the inoculated agar plates with sterile tweezers. The standard antibiotics- Gentamicin (10 \u0026micro;g / disc), BBL, 6 mm Sensi-disc- were used as the positive control, and DMSO as the negative control. The entire antibacterial assay was carried out under strict aseptic conditions. Experiments were performed in duplicate. The plates were incubated overnight at 37\u0026deg;C. A clear zone of inhibition (ZOI) was measured in millimetres with a ruler and reported as average measurements according to Clinical and Laboratory Standards Institute (CLSI) guidelines (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). Detailed figures of ZOI experiments (Figures S1 to S6) can be found in the supplementary material section.\u003c/p\u003e\u003c/div\u003e"},{"header":"3 Results","content":"\u003cp\u003eThe birch tar (BT) yield of the six samples, BT1 - BT6, varied significantly across samples, ranging from 0.06 g to 0.69 g (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). We tested the antibacterial potential of birch tar using a modified version of the Kirby-Bauer disc diffusion method. The results (see Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e) showed that birch tar was selectively active and worked against \u003cem\u003eStaphylococcus aureus\u003c/em\u003e but had no effect on \u003cem\u003eEscherichia coli\u003c/em\u003e. None of the samples produced any measurable inhibition zones for \u003cem\u003eE. coli\u003c/em\u003e (0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0 mm), indicating no antibacterial activity against this Gram-negative bacterium. In contrast, the birch tar samples showed varying inhibition levels against \u003cem\u003eS. aureus\u003c/em\u003e. The effectiveness depended on both the extraction method and the concentration used. Sample BT5 stood out, showing the highest antibacterial activity with a zone of inhibition measuring 10.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7 mm. This sample was made using the raised structure method with \u003cem\u003eBetula pendula\u003c/em\u003e bark at a concentration of 133 mg/mL. Other samples (BT1, BT2, BT4, and BT6) showed moderate activity, with inhibition zones between 7.0 and 7.5 mm. One sample, BT3, showed no activity, mirroring its lack of effect on \u003cem\u003eE. coli\u003c/em\u003e. As expected, the standard antibiotic Gentamicin (10 \u0026micro;g/disc) used as a positive control was more effective, producing clear zones of 22\u0026ndash;23 mm against both bacteria. The negative control (DMSO) showed no inhibition.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure\u0026nbsp;3\u003c/strong\u003e. \u003cstrong\u003ePhotos of sample ID BT_2024_012\u0026thinsp;\u0026minus;\u0026thinsp;01 (BT5) showing antibacterial activity against\u003c/strong\u003e \u003cstrong\u003eStaphylococcus aureus\u003c/strong\u003e \u003cstrong\u003eand\u003c/strong\u003e \u003cstrong\u003eEscherichia coli.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ea: Zone of inhibition of BT5 showing antibacterial activity against Gram-positive \u003cem\u003eStaphylococcus aureus\u003c/em\u003e ATCC 25923. b: Zone of inhibition of BT5 with no antibacterial activity against Gram-negative\u0026nbsp;\u003cem\u003eEscherichia coli\u003c/em\u003e ATCC 25922.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eDetails of samples obtained from birch tar extraction using different methods and bark species.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eOrigID\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eExtraction method\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eBark species\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSample date obtained\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSource Latitude\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSource Longitude\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSample Yield (g)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBT_2024_001\u0026ndash;01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTin Can Modern\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eBetula pubescens\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2/12/2023\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e53.54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8.75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.39\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBT_2024_005\u0026thinsp;\u0026minus;\u0026thinsp;01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCondensation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eBetula pendula\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2/27/2024\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e50.83\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e7.13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.11\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBT_2024_006\u0026thinsp;\u0026minus;\u0026thinsp;01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCondensation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eBetula pubescens\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2/27/2024\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e53.54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8.75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBT_2024_011\u0026thinsp;\u0026minus;\u0026thinsp;01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRaised Structure\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eBetula pendula\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10/3/2024\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e50.83\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e7.13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.33\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBT_2024_012\u0026thinsp;\u0026minus;\u0026thinsp;01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRaised Structure\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eBetula pendula\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10/3/2024\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e52.94\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e11.26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.69\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBT_2024_013\u0026thinsp;\u0026minus;\u0026thinsp;01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRaised Structure\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eBetula pubescens\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10/3/2024\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e53.54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8.75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.59\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2.\u0026nbsp;\u003c/strong\u003eZone of inhibition (ZOI, mm) of birch tar (BT1-6) against\u003cem\u003e\u0026nbsp;Staphylococcus aureus\u003c/em\u003e and\u003cem\u003e\u0026nbsp;Escherichia coli.\u003c/em\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cimg 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G5fF48JgYruFx0CUhoJdfZ520FOSWnF4eNjWKxjlxthmzxUNH/nxMp/eFnTRmKTrN8WWE51o9aEZQrYv/Zn1S0tLnvfUnFgs5nsj8nbgcWEwsd3CYyBKA0MfbpQhxFT1mc6JRAJTU1OI+Ax5w3iEJbQhTr1MPV0CARki1f9k2S9evMBXX33l+Q4y3JnL5VR6M8Odt2/fRj6f912Hy5DvJ+umrxeqAYy8hjasNDU15fn+eh3I+smBNpFIeF5L3navy1WXy+WwubkJq/oIwWZ6VOQJMH7PXpdgVZ7uY/bIz87Otm20YJjY2mNMYRyT6h0PwuZDB48L9I+w7RE2H9hu4Zl3uCfqR/LkCv0pFvK0Ff2pFfLUC1O8+sQuIU8tMZ86ImXoTwCS/9Nfu9Xl6q/lO8mTNOQpWfKkFFd7eon5hA0hy6/3hI1myZNz5MkrQp6+4vda6kC+p9SRPOFFfzqL1JX+P7KcdDqt2onCkfqU9vJ7mopsv+Z2Itteo+1L3ybpcqQt9DoPezwIm68TxwWqFbY9wuZju4XDHlEaCK9evQKqN1eXG6xLGqpz5+oNiwXd13J9fV29TqfT6vXJyYl6rZNycrmcmu8p5DnsAGBpz+5u5UEF7XieNKq9Nfl8Ht999x1Q/Y6iUCioqQX66/n5eZVP6vTevXtIp9PY3d2F4zj47rvv1Fn+7du3Pa+j0Sji8TgikQju3LlT0/NGwcrlMlZXVxGJRLC6ugoAeP78uZktkFwsE/RMamnPVrZJ8he0r4Y9HoTNhzrLovYJ2x5h84Ht1hADUbpy/IYyW72atVAoIJ/PIxKJwHEcdTHCH3/8gfn5eTN7S2zbRi6X86RNTEx43rdKn5JwcHCgApFyuQzHcTA/P49cLgfHcTA5OQlU1y2fz6thWhl6XF1dxerqKlzXRTQaVScCs7OzKqCXk4T9/X1ks1lYllWzbhTs8PBQXUDkVi9Cchwn9NDewsICYJykCSlDTjL8yvRLo/rata+G0c1lUfuw3epjIEoDQeZfnZ+fq94ev3lwQaLRaGCvaD2Hh4fq6mMJwFANUG/evGlmb8m9e/cwPj4OaD1Z8r4d5BZT+XxeBc/y+E5o8wpRnYdbKBQ8vcPCcRy4rquC+oODA1iW5ZkTtbu7i//93/9FIpHA0tIS0uk0/vjjD60UqkdvC1S3DRg99/WsrKzAsiysrq56Tsj0Cybkgj0pW0xNTbH3ugXmvtsJnTguUOex3UIyx+qJ+pXMmYM2b07mM6I6N1Sfu6OTuToyj0vyydxRVOfN6WU4jqM+0/+KxaJnrqj+P+Z31F/7laUvy62uo172Zcl30JedTCZ958halqXqyaw/v+8qZabT6Zp5pfryguYrkpfMNZP21+cuS7u5xn4gdW4ytzW/OWr652yny4nH477HJPMYoL8Om8/twHGBaoVtj7D5XLZbaHzEJw0NuY9o0C1w/OjPXBfZbFbNB2onuReh4zh157sSUX/p5L7bybKpc9hu4XFonobGysoKFhYWmrqd0tramhqSdl3Xd8i6XWKxGA9aRAMoGo3CcZy23ldY8LgwmNhu4bFHlKiOTCajrl4GgGQy2dab4hMREQ0zBqJERERE1BMcmiciIiKinmAgSjQk5LGe5n1WI8YjTPU/nf44VLmFk/kcZf3RnqlUCuVyuak5ucNOr2P9np7SdvIndSqPZo1UHzPYqJygdJM83rXd5NGxRESKeRk9EV1N+i2WdHJ7EfOxkfptR+R2QHJbJ7nNk55Hbl0ipDxzeeTPvF2T/qhA/dYwqN5qSb/VlnzuOE5gOUHpJvNWZ+3UybKJaDCxR5RoSEhP1MHBgSc96HZWkp7L5ZDP55FOp9UNz6PRKPTp5dIzWiwWVdrS0pLnPdX38uVLdXcGGI8KPDk58TxxKRqNYnd3V30uD3fY3d0NLCco3RSLxZBOpztytW80GkU6na7pSSei4cVAlGgI5HI5bG5uwrIs5PP5poZH5Wk/fk+ykmB1a2sLqD7uUzc7O6seFUr16XdjiMfj6lGc5XIZW1tbnikRJnm0KuqUE5Suk8e5SlunUilEIhEkEgk17SKXy6l0mSIQNh+q21E+n687NYCIhgcDUaIhcHp6img0igcPHgDVnrOw5NGoerCjk6DWsizzI2rR8fGxChz1x7E6joNIdZ6vBIt+z5UXejlh0s/OzgCtrSVPPp/H2toaLMvC8vIy5ufnEY/H8fz586by6WXLsohouDEQJbriyuUyVldXEYlE1D1R9cCgEemFC3qWtgzhBg31UnNs28b6+rqq15s3b9Y8UGF3dxezs7NIJpNYXV1VJwF6r7VZTqN0VE9Y/MTjcdXbbVmWerKY2eZh86HOsohouDAQJbriDg8PPfML4/E4HMcJPTS6sLAABPS8SRkyzOtXpl8aBVtfX1cBXC6X8wSMKysrnqd7bW5uqjbVg0D4lNMoHQAmJiY87zupm8siov7FQJToipM5nuLevXtANSAJY2VlBZZlYXV11TO3VL/gRIZmpWwxNTVVM2+UgiUSCeTzeXWLpZs3b6pbLukBvT7vdmpqCvl83nPRmV859dLF+Pg4UKf3ux2kbFkWEQ058zJ6Iro6LMtyod1mybyFTzKZdF2f2wP53XJJbuEkf3KbJ53+OW/T0xyzflE9PJttJvUut+OSNmxUTlC6KR6Pq/aXZaC6TQS9DpvPrW5r+m2/iGi48RGfRESklMtlWJYFx3F855FeRifLJqLBxKF5IiJSotEoHMdBLBYzP7q0WCzGIJSIPNgjSkREREQ9wR5RIiIiIuoJBqJERERE1BMMRImuOLn9j3kLINu2Vbr+GMZcLqfS9PtMBpWj59cf5Wgql8uBj6i8jKmpqaYeWUpERP2Dc0SJrjDbtjE3N6feW5alHtmZyWTUk5agPf3GsiwUi0W8evUKq6urcBwH5+fnvuXYto2XL19ic3NT3aPS75DSyaulO1k2ERF1FntEia6wly9fqicqwXjU4snJieeJS9Fo1PMMenlc5O7ubmA5o6OjnueMyxOWTLFYDOl0uiOBYjQaRTqd9txgn4iIBgMDUaIrTIJEVB/DKYFiuVzG1tYWIpFI4HD56Oioeh1UTjQaRblcRiQSUe9Ntm3DcRwV2KZSKUQiESQSCSQSCTUFQNJleD9sPlSD5nw+z8eJEhENGAaiREPi+PhYBZSHh4cq3XEcRCIRlMtlFSz6PVde6OWgGnxKT+nW1lZNMHh2dgZoga3eg7q2tgbLsrC8vIz5+XnE43E8f/68qXx62bIsIiIaDAxEiYaAbdtYX19XPZY3b95UQ+3pdBqoDsHPzs4imUxidXUVlmUB2hC9Xzk6Ga43g8HT01PPexGPx9Vz6C3LwtLSEmBMH2gmH+osi4iI+hMDUaIhsL6+rgK4XC7nCSRXVlZUMIpqT6TrumoIXoJA+JSji0ajKnjVTUxMmEkd081lERHR5TEQJbri5Gp2ucXSzZs31a2Y9GH0lZUV9Xpqagr5fB77+/sqza+cRCKh5pjKLZQkUBXj4+MAgPPzc096O0nZsiwiIhoMDESJrjAJHnXRaFQNt8/NzSESiaieRLkQKBaLeW7DFFTOwsKCmmMai8XUraF0s7OziMfjat6pXN2ez+eRyWTgOA4cx0Emk1HLyGQyofOhOqfV7L0lIqL+x/uIElHHdfJen50sm4iIOos9okTUcdFoFI7jIBaLmR9dWiwWYxBKRDSg2CNKRERERD3BHlEiIiIi6gkGokRERETUEwxEiYiIiKgnGIgSERERUU8wECUiIiKinmAgSkREREQ9wUCUiIiIiHqCgSgRERER9QQDUSIiIiLqCQaiRERERNQTDESJiIiIqCcYiBIRERFRTzAQJSIiIqKeYCBKRERERD3BQJSIiIiIeoKBKBERERH1RMR1XddMDCMSiZhJRERERNTHWgz7OqblQPSvv/4yk4iIiIioiz799FMzaaBwaJ6IiIiIeqIjgejDhw9x48aNmr9CoQAA2NjYqPlMz1OpVNT7L774wiy+5y67fvXs7OyovKVSSaWXSiXf9KD8olQqharDoPL90nvVPoVCoea7wPg+lUrF8z8EVTf16qiddSjb5MOHD82PlDB5hhHbYTDpvwlh0lsRpq3C5KEPuK/1j44Eos+ePcOdO3cAAHt7e9jb2wMAPHnyRDX43t4ejo6OAAAzMzM4OjrC06dPAQBjY2M4OjrC+Pi4KrOfXHb9gpRKJfzwww/q/b///W/1+v79+9je3sb29jbu37/fMH+9ND9+5Qel96J9KpUKNjc3cXR0hL29Pc93/Pbbb1U7fPvtt57/G3aFQgFHR0fqT7ZbU9g6rFQqdU+mSqUS3rx5g6OjI3z22WfY2dkxs4TKM6zYDoNnZ2cH33zzjdq/JNAISvfD9uw+7mv9oyOBqGlsbAwzMzMAgIuLCzx+/BhjY2NmNsRiMcRiMTO577Vr/fb391XAAABnZ2dANZgAgJGREYyMjKi0oPxiZ2enJs1PUPlB6b3w999/48svvwSq9X3nzh0UCgWUSiV8/PHHGBsbw9jYGG7duuXpLR12ftubuW22sw739/fx+eefAwDu3r2LX375xcwSKs8wYjsMprt372J6ehoA8PXXX+P169d101sRpq3C5KEPuK/1l64EopVKBa9fv8b4+LjaMa+Sdq3f48eP1euZmRkV3L5//17L9cH79+8D8+vC9FoGlR+U3gvv37/H+fm5ej86Oor379/j7du3+Oyzzzzpb9++Ve/pH5VKBaOjo2ZyW+vwzz//xLVr1zxp5rBXmDzDiO0w+MbGxnyPuUHpYYVpqzB56APua/2l44Ho4uIiFhcXMT4+jl9//dX8eOB1av3evXvnCTRNelAGn/wbGxu4e/euJ08zzPJFUHqnXb9+Hbu7u+r9+fk5PvroI08equ/333/H9evXzeRQZC7V4uIinjx5ot632otArWE79LdSqaRGbsKksz37F9umezoeiMpcybOzsyvZiJ1Yv1KphFQqVTOEqtN7tsz8pVIJn3zyiZa7eX49Z6iT3mnT09O4c+eOOhjs7u7WnGFSfefn5y332MsUkL29PTx9+lS9b7U8ag3bob/t7+/7dgAEpbM9+xfbpns6HoiKR48eAdUu8auonev3008/qbl9hULBt+dPTzPz7+/vqzO4s7MznJ2dYWNjQ/tvr6Dyg9KD6FeI1lteqx4/foyjoyNsb29jZmYGY2NjuH79Ot68eaPyvHnzpuVev2HVzjq8desW/v77b0AbejJPqMLkGUZsh8G2s7ODRCJhJgemNyNMW4XJQx9wX+svXQtEZXJuvUBmkLVr/R4+fIjXr1+rgO7atWsqyLy4uMDFxQWgXYTil18CtqPqle3j4+N1h/mDyg9KD/Ls2TO13HrLu4xSqYT79+/j2bNnQLWn9N27d6hUKqhUKnj37h3PWH0UCoXAXvIwdRh2mCqRSOC3334DqlMB/IYjw+QZRmyHwSUXcU5PT6NSqairooPSBduzN7iv9ZeOPFlJgiPTo0eP1PBEpVLB4uKi+kyfY2l+huoQeL+cQVx2/YL4lXtUvSJeAjAA2N7exvT0dN38Qu7z2WjZfuUHpZvrhi60T6FQwJMnTzAzM6OCUKF/x05/j0G1sbHhOTmQNpQ2bWcdbmxsYHd319NWOzs7+OWXX9R26JeH2rst+9Ux26H9pA51e3t7+Pnnn33TW21Tv7Zie7buKu1rg/5kpY4EokRERETUeYMeiHZtaJ6IiIiISMce0S7zG9YWlx0eqKdXyyUiIqLOGfQeUQaiRERERANq0ANRDs0TERERUU8wEG1RqVSquZ1DpVJRaXK1OhERERH5YyDaovv372N7exvb29vqFhBjY2Pq3p1EREREVB8D0RbITYpHRkYwMjLiSSMiIiKicBiItuD9+/dmkm8aEREREQVjINom5+fnZhIRERER1cFAtE1GR0fNJCIiIiKqg4FoCz766CMzyTeNiIiIiIIxEG1BLBYDAFxcXODi4sKTRkRERETh8MlKLSqVSuq2Tdvb25ienvZ9jCYfn0lERESdMuhPVmIgSkRERDSgBj0Q5dA8EREREfVEyz2iRERERESXwR5RIiIiIuqJjgSiiUQCkUik5i+XywEAUqlUzWdmnkwmo9Js2zaW0FvtWL8gQett27ZvelB+Yds2pqamzOQaQeWHSY9EIshkMuqzTpP6b1SX9IHeTpFIBOVy2cyCcrlc9/NmyDaZSCTMj5QweYYR22Ew6b8JOv34zPbsL9zX+kdHAtH9/X0kk0kAgOM4cBwHALC8vKwa3HEcyKyAeDwO13WRzWaBapCzurqqyrt375563Q8uu35B6q333NwcisUiisUi5ubmGuavl+bHr/x66a9evVKvAeDOnTue950SiUQQjUbhui6WlpbMj8mQy+Xguq76k+3WFIvF1LZc71Zk5XK57gmAbds4ODiA67pYWFjwPUEJk2dYsR0GTyaTwdramtq/JNCQ3wLXdZFOp/H9998b//kPtmf3cV/rI26HJJNJF4DrOI7ruq4bj8ddAG6xWPTkA+DG43FPWjKZ9Hzewa/ZssusX5Cg9c5ms2pZjuO4ANxsNhuYX6TTaReAa1mWJ90UVH5Qumt8126Jx+NuOp02k6kJfu1WLBY922gymazZjoXjOGob8JNMJj2f+217YfIMI7bD4JPjpLwWxWKx7rGL7dld3Nf6S0d6RE3lchn5fB6WZWF2dtb8uMbm5qZ6HY/HEY/HPZ/3m2bXL0jQep+enmq5Pjg9PQ3Mr7Msy0yqEVR+UHq5XMbW1hYikUioYf92KJfLOD4+xsHBASKRCFKplJmFGiiXy5icnDST8erVKywsLKj3k5OTNT3eYRUKBdy8edOTZg57hckzjNgOgy8ajapjbjQaVekvX77EysqKlrM5YdoqTB76gPtaf+l4IGpZlvo7Pj42P27o+PjYE3D1m8uuX5BG631ycuJ5b+ZPpVKXOvCZ5YuTkxMcHh6q947jtGWOTSOHh4eYmppSQ2BbW1ueOavU2O7uLm7fvm0mhyJzqSzLwvLysnrPNugutkN/s20bDx48UO/L1XmIW1tbvsOxbM/+xbbpno4HojJXUgKWZhrRtm2sr697ziz7zWXWL0iY9dZ7tsz8tm1jfn5ey908v54zVNNv3rwJtzrnMJ1OA9Ugp5NOT0+xsLCgepyTyWTLZ7DD6uTkpOUee2lvx3GQzWbV+1bLo9awHfqb2fMp89nT6TSeP3/uyQu2Z19j23RPxwNRIQFLM8HD+vq6uiCl3mThftDK+gUx13tiYsLM4kkz8798+VKdwclk7HpD2UHlB6XrAfLKyopad/3K0XrLa8XExISnl3ZyctL3+1Hzbt++jYODA/X+4OCg5Z7TWCymesyll9w8oQqTZxixHQZbJpPBV199ZSYD1ePkZYRpqzB56APua33GnDTaLubFPJZleS52EQi4mEcu/pE/feJ3P7js+gUJWm9UL4QqFotqMny9/MKyrFATo4PK90uXi6Bkcne9SfjtZNa3ua4ULFu9+CyI1KfjOL7bi76N6X/mBH/9IoB0Ou27bYTJM6zYDoMpm82q+nMcp6Yui8Wi74WCZjuyPbuH+1r/6EggagZH8qdXvlO9ulD+9A3B7//7id/3a2b9gviVKyQQhLYj1MsvwgaifuUHpetp8Am+O0VfbreWeVWYP4Kyffq16WUDfDlJ00/A0um0Zzv0y0Nsh0Ekdaj/STAq79tRv35txfZsHfe1/sFHfBIRERFRT3RtjigRERERkY6BaJfJ7Tz8/jp5C6ReLZeIiIgoCIfmiYiIiKgn2CNKRERERD3BQLRFtm2roW25ib0+/N2tR18SERERDSoOzbcoEomgWCwCAObm5qBXowSh7XzkJxEREdFVwx7RFshTnkZHRzE6OupJIyIiIqJwGIi24PT01EzyTSMiIiKiYAxE20R/DjoRERERNcZAtE0mJyfNJCIiIiKqg4FoCyYmJswk3zQiIiIiCsar5lvEq+aJiIiILoeBaIts28bc3BwAoFgsYnZ2FuVyGZZlefI5joNoNOpJIyIiIiIGokRERETUI5wjSkREREQ9wUCUiIiIiHqi5aH5v/76y0wiIiIioi769NNPzaSBwh5RIiIiIuqJjgSiDx8+xI0bN2r+CoUCAGBjY6PmMzNPoVBQaTs7O8YSeqsd6xdkZ2dH5S2VSiq9VCr5pgflF6VSCV988YWZXCOo/KB0Ieu6sbFhftQxOzs7nm2iUqmo71ipVFS6Xu/mZ8MkqH50YfKEJdvkw4cPzY+UMHmGEdthMOm/CTr9+Mz27C/c1/pHRwLRZ8+e4c6dOwCAvb097O3tAQCePHmiGnxvbw9HR0cAgJmZGRwdHeHp06dANfj5v//7PxwdHWFmZgY//PCDKrsfXHb9gpRKJc+6/vvf/1av79+/j+3tbWxvb+P+/fsN89dL8+NXfr10VAO93d1dHB0d4fHjx57POsncHr799lvVDt9++y1QPZE5OjpSf9Jew8ivfkxh8qB68K53MlUqlfDmzRscHR3hs88+8z2JDJNnWLEdBs/Ozg6++eYbdZyRQEN+C46OjvDo0SP8/PPPxn/+g+3ZfdzX+kdHAlHT2NgYZmZmAAAXFxd4/PgxxsbGzGyIxWKIxWIYGRlRgc3r16/V//arZtcvyP7+vgqcAODs7AyoBlUAMDIygpGREZUWlF/s7OzUpPkJKj8oHdUeAAmwu2ljYwOPHj1S70ulEj7++GOMjY1hbGwMt27dQqlU8q1nvza56oLqp9k8Ye3v7+Pzzz8HANy9exe//PKLmSVUnmHEdhhMd+/exfT0NADg66+/xuvXrz2fAcD169cxOjqq0psVpq3C5KEPuK/1l64EopVKBa9fv8b4+LjaYesZGxtT3ebyvp81u35B9F7FmZkZFdy+f/9ey/XB+/fvA/PrxsfHzaQaQeUHpZdKJbx+/RqvX7/u6lBDqVTCJ5984kl7+/YtPvvsM/V+dHQUb9++9eSpVCqX+hEYZGHqJ0yesP78809cu3bNk2YOe4XJM4zYDoNvbGxMHXP13639/X0VlLYiTFuFyUMfcF/rLx0PRBcXF7G4uIjx8XH8+uuv5seBxsbGVG/b7u5uy2crndbq+jXy7t27usPd5+fnnvdm/o2NjUsd+Mzyxfn5udpht7e38fTpU7x+/bru0EW77O/v+/Z0NvL777/j+vXrZjI1QeZSLS4u4smTJ+p9v+6XVxXbob+VSiV8+eWX6r10qOzu7voOx7I9+xfbpns6HojKXMmzs7OWGlHmX/73v/81P+oLl10/P6VSCalUqm5PsN7DZ+b36zlsVlAPop4+MjKizvL8ek/bqVAo4OuvvzaTQzk/P79UTzV9mOd2dHSEvb09PH36VL1nvXYX26G/mT2f0qHy6NEj3+FYtmf/Ytt0T8cDUSHz+prt/taHOvpZq+vn56efflI9f4VCAR999JGZxZNm5t/f31dncGdnZzg7O6t7VXtQ+UHp4uLiwvOZfuVoveW14rfffsPi4iJu3LiBH374AT/88AM2NjZw/fp1vHnzRuV78+YNez81YeonTJ6wbt26hb///hvQhp7ME6oweYYR22Gw7ezsIJFImMmANle0VWHaKkwe+oD7Wn/pWiAqZ4N+wY3p4cOH6pZD0mitDMl2UzPrV8/Dhw/V3MsbN27g2rVrat0vLi5U8CdpfvkfP36szt7Gx8cxPj5ed5g/qPyg9H/9619ANeiW9OvXr+PZs2dqufWW1wq97EePHuHRo0d4/Pgxpqen8e7dO1QqFVQqFbx7985zxlooFC7dOzzIGtVP2Dxhh6kSiQR+++03oDolQh+mbCbPMGI7DC6ZmjQ9PY1KpVIzDF8qlXDr1i1PGtiePcN9rb905MlKEhyZHj16pM4MK5UKFhcX1Wf6HMudnR11i552z71sh8uuXxC/co+q82RLpZK6fdL29jamp6fr5hcS0Ddatl/59dILhQKePHkCGOvdDXKQl2Xq33Fvb89zprmxsdH2oHjQ+NWPbJ/Spn55WrWxsYHd3V3MzMzg2bNnQLXNfvnlF7Ud+uUh/7ZqlV8dsx3aT+pQt7e3h99//139jrWjfv3aiu3Zuqu0rw36k5U6EogSERERUecNeiDataF5IiIiIiIde0S7zByy1112eKCeXi2XiIiIOmfQe0QZiBIRERENqEEPRDk0T0REREQ9wUC0RaVSqeZ2DpXqUzRu3LihrlYnIiIiIn8MRFt0//59bG9vY3t7W90CYqz6FI1BuAE/ERERUa8xEG2B3Lx4ZGQEIyMjnjQiIiIiCoeBaAv8nqvul0ZEREREwRiItsn5+bmZRERERER1MBBtk9HRUTOJiIiIiOpgINqCjz76yEzyTSMiIiKiYAxEWxCLxQAAFxcXuLi48KQRERERUTh8slKLSqWSum3T9vY2pqenfR+jycdnEhERUacM+pOVGIgSERERDahBD0Q5NE9EREREPcFAlIiIiIh6ouWheSIiIiKiy2CPKBERERH1REcC0UQigUgkUvOXy+UAAKlUquYzM4+wbRtTU1OetF5r5/qZMpmMymvbtkq3bds3PSi/CFt/fuWXy2WVppcRlN5p+rqWy2WVrn8fPZ0+kLppVEdTU1Mqz2VIOyUSCfMjAEAul/N8n6B8w6id23KjdkDIPNSY/psQJr0VYdoqTB76gPta/+hIILq/v49kMgkAcBwHjuMAAJaXl1WDO44DmRUQj8fhui6y2axWygf37t0zk3quneuns20bq6ur6r2+7nNzcygWiygWi5ibm2uYv16aH7/yo9EoXNeFZVmevEHpnST16rou0uk0vv/+e/VZLBZT7cD7uXrlcjm4rqv+ZLs15XI5FAoFlS9IuVyuezJl2zYODg7gui4WFhaQyWTMLEC1HRstaxiF3Zbb0Q5h8lBjmUwGa2trav+SQCMo3Q/bs/u4r/URt0OSyaQLwHUcx3Vd143H4y4At1gsevIBcOPxuCdNpNNpF4BrWZb5Uc+1Y/1MyWRSvQbgSvNks1m1LMdxXABuNpsNzC/C1l9Q+cKyLN8ygtI7QerZdV23WCy66XRavdbrN5lM1rQB/UPfZnSWZfluvybHcTzbhimZTNZsO/UUi8W65Q2TZrbldrRDmDzUHDl+moLSBduzu7iv9ZeO9IiayuUy8vk8LMvC7Oys+XFd3ex1a9Vl1k+3ubmpXsfjccTjcQDA6empluuD09PTwPy6MPUXVH4/iUaj6vXLly+xsrICAHj16hUWFhbUZ5OTk3j16pV6T/8ol8uYnJw0kwEAhUIBjuNgbm6u7tl/I4VCATdv3vSk1Rv2evXqVU3+YdXObTlMO4TJQ82JRqO+x9yg9LDCtFWYPPQB97X+0vFA1LIs9Xd8fGx+HCiVSqlgo5+1un6NHB8fewJN08nJiee9mf+y9WeW3w/K1Tk9W1tbHNpowe7uLm7fvm0mA9Ufymg0CsdxsLa2Zn6s5lJZloXl5WX13m9ecjMODg48JxlUX6fagdrDtm08ePDATA5MZ3v2L7ZN93Q8EJW5ko7jhG5E27YxPz9vJvelVtavEdu2sb6+XvcHWu/ZMvO3o/6Ces56SeamptNpPH/+3PyYGjg5OWnYYx+NRn0vQHOr8zkdx0E2m1XvG5VXT7lc9vRKUGOdaAdqH320Jkw627N/sW26p+OBqEin00C1S7yRly9fqjMQmUycSqXMbH2lmfVrZH19HUtLS0D1IpKJiQkziyfNzN9s/TUqPyz9CtF6y7ss/YB++/ZtHBwcqPcHBweBvX4UTr0ToEZisRgODw8BbegpqLzDw0O2laad23KYdgiTh8LLZDL46quvzOTA9GaEaasweegD7mt9xpw02i7mxTxyMYQ56RcNLubp5gUxzWjX+pnkoif5k/JRvZCkWCx6Jr0H5Rdh6y+ofLdOGUHpnVYsFj0X3ViWpS606sX3GQTZbLZm2/QTlE/fxvQ/c4K/fhFAOp1WF5X5aWa/GBaNtmWz/i/TDmHyUDjZbFbVn+M46nVQujDbke3ZPdzX+kdHAlEzOJI/vfLlKkL589sQ3B4GO/W0c/10fuUKCRCh7Qj18ouw9edXvrkOMK6sN9M7KV29AwB8Anv9u3f6ewwq82p5acNi9ap1qT8zXyvkJE1vp3Q6XbMdtmNZV007t+Uw7eCXh5ojdWgeD4PSW+XXVmzP1nFf6x98xCcRERER9UTX5ogSEREREekYiHaZ3ILI76+T9xXr1XKJiIiIgnBonoiIiIh6gj2iRERERNQTDERbZNu2GtqWm9jrw99+NwUnIiIion9waL5FkUgExWIRADA3Nwe9GiUIbecjP4mIiIiuGvaItiCXywEARkdHMTo66kkjIiIionAYiLbg9PTUTPJNIyIiIqJgDETb5OTkxEwiIiIiojoYiLbJ5OSkmUREREREdTAQbcHExISZ5JtGRERERMF41XyLeNU8ERER0eUwEG2RbduYm5sDABSLRczOzqJcLsOyLE8+x3EQjUY9aURERETEQJSIiIiIeoRzRImIiIioJ1ruEf3rr7/MJCIiIiLqok8//dRMGijsESUiIiKinuhIIPrw4UPcuHGj5q9QKAAANjY2aj4z85RKJU/6zs6OsZTeacf6BdnZ2VF5S6WSStfrQ08Pyi9KpRK++OILM7mGX/mVSkWlmWU0Wm6nbGxsYGNjw5Omf5dKpdIwfRj51ZvevvXq54svvlD5LkPa4+HDh+ZHAIBCoaCWUy/fVRa2TcJoVN8ImYca038TdO08BoVpqzB56APua/2jI4Hos2fPcOfOHQDA3t4e9vb2AABPnjxRDb63t4ejoyMAwMzMDI6OjvD06VNVxtu3b9VrAPjXv/7led9L7Vg/P6VSCT/88IN6/+9//1u9vn//Pra3t7G9vY379+83zF8vzY9f+WNjYzg6OsL4+Lgnb5jldsLGxgZ2d3c9aVLfR0dHePToEX7++ee66cPIr94A4Ntvv1Xb77fffmt+DFSDwx9//BFHR0dqe/ZTqVTqnmSVSiW8efMGR0dH+OyzzwJPLGU59ZZ1lYVpE7SpvsPkocZ2dnbwzTff4OjoCHfu3FGBRjPHILZn93Ff6x8dCURNY2NjmJmZAQBcXFzg8ePHGBsbM7MhFoshFosBAM7Pzz0/Sn75+0Ur6+dnf3/f8yN8dnYGVIMBABgZGcHIyIhKC8ovdnZ2atL8BJUfpNFyO+Xx48d49OiRmYy7d+8CAK5fv47R0dGG6cPGr95KpRI+/vhjjI2NYWxsDLdu3fLt2d7c3MTi4qLvZ83Y39/H559/DlTb5ZdffjGzePaNUqmk8g+LsG0SRpj6DpOHGrt79y6mp6cBAF9//TVev37t+QxtOAaFaasweegD7mv9pSuBaKVSwevXrzE+Pq522HoqlQp2d3dxw2dIuB81u35BHj9+rF7PzMyo4Pb9+/darg/ev38fmF9n9mb6CSo/SJjldose8O/v76sDf1A6ffD27Vt89tln6v3o6GjNKAQA/Pjjj9jb28P9+/frnpw08ueff+LatWuetHrDYW/fvq3Jf9WFbZMwwtR3mDzUnLGxMXXMbecxKExbhclDH3Bf6y8dD0QXFxexuLiI8fFx/Prrr+bHvv7++2/1+uzsrC1zODqllfUL4927d56Az3R+fu55b+bf2Ni41IHPLD+IudxeqFTn+uzu7nqGPILSKTzpMdjb28Pm5qb5sZpjtbi4iCdPnqj3rfYuiDdv3viOKgy7TtU3tUepVMKXX36p3jc6BrE9+xfbpns6HojKXEkJKMM04rVr19TQrwwp/v7772a2vtDK+jVSKpWQSqXq/hDrwzxm/lKphE8++UTL3bwww0jmcntlrDqX9dGjR54hj6B0at7Y2Bg+/vhjM1ntp3t7e3j69Kl6f5mRgUql4umtoH90or6pfcyez0bHILZn/2LbdE/HA1EhAWWY7m89sLl7927N/LZ+1Mz6NfLTTz+p+XKFQgEfffSRmcWTZubf399XZ3BnZ2c4OzuruWJa16j8IOZy9StH6y2vU4J6gIPSh9n169fx5s0b9f7Nmze4fv26J4/pMicct27dUiMdMroRVN7ff//d8LtcRa20SZAw9R0mD4W3s7ODRCJhJgNtOAaFaasweegD7mv9pWuBqJwNhglw5DYHeu/iZXfkTmtm/ep5+PAhXr9+rQK6a9euqWDv4uICFxcXgHZhh1/+x48fq7O38fFxjI+P1x0+r1d+EL/lPnv2TC233vI6pVQq4datW2ZyYPowm56exrt371CpVFCpVPDu3bu6Z/qFQsG3lz3s8FUikcBvv/0GVEc39OFL02+//Vb3u1xVYdqknfUdJg+FI/Onp6enUalUaobhg45BbM/e4L7WXzryZCUJUkyPHj1SAWWlUsHi4qL6TJ9jWSqV1C2EAODp06cNA6Nuuuz6BfEr96h6ZbpeJ9vb25ienq6bX8jFXo2W7Ve+uQ6oTkXY2NhouNxO2NnZUbeNunPnDh4/fuxJm5mZwbNnz2ry6unDyK/eYLT53t4exsbGVJtvb2/jv//9L548eVLzf62S20iZ7fTLL794ts+NjY1LL2tQ+bVJq8LUt18eao7f7dH29vbw+++/t/UY5NdWbM/WXaV9bdCfrNSRQJSIiIiIOm/QA9GuDc0TEREREenYI9plfsPd4rLDA/X0arlERETUOYPeI8pAlIiIiGhADXogyqF5IiIiIuoJBqItKpVKNbdzqFSfonFjQB5NSkRERNRLDERbdP/+fWxvb2N7e1vdAkKeohHm+e5EREREw46BaAvk5sUjIyMYGRnxpBERERFROAxEW/D+/XszyTeNiIiIiIIxEG2T8/NzM4mIiIiI6mAg2iajo6NmEhERERHVwUC0BR999JGZ5JtGRERERMEYiLYgFosBAC4uLnBxceFJIyIiIqJw+GSlFpVKJXXbpu3tbUxPT/s+RpOPzyQiIqJOGfQnKzEQJSIiIhpQgx6IcmieiIiIiHqCgSgRERER9UTLQ/NERERERJfBHlEiIiIi6omOBKKJRAKRSKTmL5fLAQBSqVTNZ2YeIXlTqZQnvZfauX6mTCaj8tq2rdJt2/ZND8ovbNvG1NSUmVzDr/xyuazSzDJyuZz6LJPJeD7rpKmpKbVcoX+XRvU7jKRu5K9cLptZPG3t93kzZJtMJBLmR0qYPMOI7TCY9N8EnX58Znv2F+5r/aMjgej+/j6SySQAwHEcOI4DAFheXlYN7jgOZFZAPB6H67rIZrNaKR9+QLe2tuC6LjY3Nz2f9VK71s9k2zZWV1fV+3v37qnXc3NzKBaLKBaLmJuba5i/Xpofv/Kj0Shc14VlWZ68tm3jjz/+gOu6iMfjnu/QSblcDoVCAa7rqrpFtd6lHZaXlz3/M+xyuZyqL9d11XZrisViqg7r3RO3XC7XDfZt28bBwQFc18XCwoLvSUqYPMOK7TB4MpkM1tbW1P4lgYb8Friui3Q6je+//974z3+wPbuP+1ofcTskmUy6AFzHcVzXdd14PO4CcIvFoicfADcej3vS3Gp+v/R+cdn185NMJtVrAK40TzabVctyHMcF4Gaz2cD8Ip1OuwBcy7I86aag8oVlWZ4yZJ3dJtfvsizLqqljx3E8382yLM/3Iy99mxHFYtHThslksmY7Fo7jeLYNUzKZrNl2TGHyDCO2w+CT46e8FsVi0U2n01pOL7Znd3Ff6y8d6RE1lctl5PN5WJaF2dlZ8+Matm0jn88jn88PRFd2s+sXRO/1jcfjiMfjAIDT01Mt1wenp6eB+XVmb6afoPKDRKNRNawh77uhUCjAcRzMzc2pM1RZdiaTQblcRiwW69r3GTTlchmTk5NmMl69eoWFhQX1fnJyEq9evfLkCatQKODmzZueNHPYK0yeYcR2GHzRaFQdc/Xj0MuXL7GysqLlbE6YtgqThz7gvtZfOh6IWpal/o6Pj82PfckGUSwWkc1mkc/n63aN91Ir6xfG8fFx3ekIJycnnvdm/lQqdakDn1m+SYbtAWBra8t3fmq7RaNRRKNROI6DtbU1lV4oFLC6uopYLFa3zobd7u4ubt++bSaHInOpLMvC8vKyet+Ndqd/sB36m23bePDggXovJ+xbW1u+w7Fsz/7FtumejgeiMlfScZymG3F0dFSdRdTroeuly6xfENu2sb6+XrdnT+/ZMvPbto35+Xktd/P8es78yPzYs7Mz86OOiUajnounDg8PUSwW4ThOX13U1m9OTk5a7rF3q3NMHcdBNptV71stj1rDduhvZs+nnLCn02k8f/7ckxdsz77GtumejgeiIp1OA1pvZxjn5+dmUt9qZf2CrK+vY2lpCahebDIxMWFm8aSZ+V++fKnO4GQydr0ArVH59ehDUfqVo/WW1w4SdJfLZbx48QKzs7NwXReFQqEtJwPD5Pbt2zg4OFDvDw4OWu45jcViODw8BLShJ/OEKkyeYcR2GGyZTAZfffWVmQwAlxqdQsi2CpOHPuC+1mfMSaPtYl7MIxeamJN+/S52kQnf6XTaLRaLNReo9IPLrF89ctGT/En5UgdSH43yC/NCoyBB5bs+ZcTjcfXevFioG7LZrKpn8/taltV320o/0OvMj1W9yCuoPfVtTP8z61q/CCCdTvteoBEmz7BiOwymbDar6s9xnJq6LBaLvhcKmu3I9uwe7mv9oyOBqBkcyZ9e+RJsyp+5IWSrV3Kb/9cP2rF+fvzKFRJwQdsR6uUXZhAZxK98cx1QDXTT1avxEXK92kHfHswDuv59zM/oA7NepG2lrfX2N09mmiUnafoJWDqd9mwrfnmI7TCIpA71P/M42Y769WsrtmfruK/1Dz7ik4iIiIh6omtzRImIiIiIdAxEu0xu5+H318n7ivVquURERERBODRPRERERD3BHlEiIiIi6gkGoi2ybVsNbct9K/Xhb/2G60RERERUi0PzLYpEIigWiwCAubk56NUoQWg7H/lJREREdNWwR7QF8tz70dFRjI6OetKIiIiIKBwGoi3we+69XxoRERERBWMg2iYnJydmEhERERHVwUC0TSYnJ80kIiIiIqqDgWgLJiYmzCTfNCIiIiIKxqvmW8Sr5omIiIguh4Foi2zbxtzcHACgWCxidnYW5XIZlmV58jmOg2g06kkjIiIiIgaiRERERNQjnCNKRERERD3Rco9oJBIxk4iIiIioi1oM4/pGy4EoEREREdFlcGieiIiIiHqCgSgRERER9QQDUSIiIiLqCQaiRERERNQTDESJiIiIqCcYiBIRERFRTzAQJSIiIqKeYCBKRERERD3BQJSIiIiIeoKBKBERERH1BANRIiIiIuoJBqJERERE1BMMRImIiIioJxiIEhEREVFPMBAlIiIiop5gIEpEREREPcFAlIiIiIh64v8DuNgHaof8uMAAAAAASUVORK5CYII=\"\u003e\u003c/p\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eHere, we discuss our samples in direct comparison with samples obtained by other studies to establish differences related to the production method and birch species and consider the implications of our results for Middle Palaeolithic healthcare practices.\u003c/p\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e4.1 Comparative analysis\u003c/h2\u003e\u003cp\u003eOur results show that birch tar has a selective antibacterial effect, working specifically against Gram-positive bacteria like \u003cem\u003eStaphylococcus aureus\u003c/em\u003e. The inhibition zones we recorded ranged from 7.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0 mm to 10.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7 mm, suggesting a moderate but clear activity (see Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThis pattern aligns with evidence from other plant-based antimicrobials, which generally exhibit more potent effects against Gram-positive bacteria. The primary explanation for this is their structural variation: Gram-positive bacteria show a thicker peptidoglycan layer, whereas Gram-negative bacteria have an additional outer membrane that can prevent antimicrobial entry (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eOur findings not only reinforce what past scientific studies have reported, but they also echo traditional knowledge. Birch tar has long been recognised for its antiseptic properties, particularly in treating skin infections, and our study adds new evidence to support its potential use in modern and historical healthcare practices. Similarly, the outcomes align with the traditional practices of the L\u0026rsquo;nu (Mi\u0026rsquo;kmaq) people, who have used birch bark extract to address skin infections commonly caused by \u003cem\u003eS. aureus\u003c/em\u003e (\u003cspan additionalcitationids=\"CR42 CR43 CR44\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eEarlier research has shown that birch tar contains a range of phenolic derivatives, such as catechols and guaiacols (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e), which are likely responsible for its antibacterial and antifungal effect (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e). Indeed, Richert et al. (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e) reported that phenolic compounds in birch tar contribute substantially to its antimicrobial activity, with pronounced effects on Gram-positive bacteria. The selective inhibition of \u003cem\u003eS. aureus\u003c/em\u003e found in our research thus aligns well with the known antimicrobial capabilities of phenols (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e). This relationship has also been investigated by Agarwal et al. (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e), who assessed how polyphenolic content relates to antibacterial effects by comparing bark extracts of wild cherry, larch, and sweet chestnut against \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e. Their bacterial growth curves indicated that the extracts did not suppress \u003cem\u003eE. coli\u003c/em\u003e; in fact, bacterial proliferation was even enhanced. In contrast, \u003cem\u003eS. aureus\u003c/em\u003e was strongly inhibited. Their sweet chestnut extract was most effective which they linked to its high antioxidant potential and highest total phenol content compared to wild cherry and European larch. None of the samples showed measurable inhibition of \u003cem\u003eE. coli\u003c/em\u003e (0\u0026thinsp;\u0026plusmn;\u0026thinsp;0 mm), implying limited efficacy against Gram-negative species, likely due to lower phenolic levels and the protective barriers of their outer membrane. This selectivity may also be a result of the distinctions in cell wall structures between Gram-positive and Gram-negative bacteria, reflecting variations in cell surface (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e) and the outer membrane structure (53).\u003c/p\u003e\u003cp\u003eHitherto unpublished data by Kaliaperumal et al. (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e), who have assessed the antibacterial properties of industrially produced maskwio\u0026rsquo;mi made from \u003cem\u003eBetula papyrifera\u003c/em\u003e, compares well to the results presented here. The antibacterial activity of their extracts TE1 and TE2 revealed higher inhibition zones against \u003cem\u003eS. aureus\u003c/em\u003e strains (18\u0026ndash;20 mm for TE1 and 11\u0026ndash;12 mm for TE2) compared to our BT samples, which ranged from 7.0 to 10.5 mm. The higher inhibition zones of their extracts may suggest that TE1 and TE2, particularly TE1, possess more potent bioactive compounds than our BT samples. Kaliaperumal et al. (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e) identified over 60 bioactive compounds, including phenols, monoterpenes, and triterpenes, in the chemical analyses (GC-MS) results of their extracts. Based on the GC-MS analysis, the authors attributed the higher bioactivity of TE1 to its elevated triterpene concentration, including betulin and lupeol derivatives, which are known for their antimicrobial effects (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn contrast, their extract TE2 exhibited lower activity, potentially due to its higher naphthalene derivative content, which may be less effective against Gram-positive bacteria. This may explain the highest antibacterial activity against \u003cem\u003eS. aureus\u003c/em\u003e recorded for our sample BT5 (10.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7 mm ZOI) in comparison with other samples BT1, BT2, BT4, and BT6 (7.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0 to 7.5 -\u0026plusmn; 0.7 mm) in our study, which may suggest that BT5 contains higher concentrations of bioactive compounds than others. Phenolic compounds appear to be key contributors to the antimicrobial activity of birch tar, although volatile compounds may also have an effect (\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e). Further analysis of GC-MS on the birch tar samples would be ideal to validate the compounds present in our samples. Just like our samples, the samples TE1 and TE2 extracts displayed weak activity against Gram-negative bacteria, with minimal inhibition against \u003cem\u003eE. coli\u003c/em\u003e, including \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e, and \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eThis further supports the hypothesis that the antimicrobial compounds primarily target Gram-positive bacterial species. Several previous studies have examined the antibacterial properties of related extracts. Acquaviva et al. (\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e) reported that the extract of \u003cem\u003eBetula aetnensis\u003c/em\u003e leaves inhibited \u003cem\u003eS. aureus\u003c/em\u003e but had a lower impact on Gram‑negative bacteria. Similarly, Emrich et al. (\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e) reported that water-based extracts of \u003cem\u003eBetula pendula\u003c/em\u003e were highly effective against \u003cem\u003eS. epidermidis\u003c/em\u003e and MRSA. Vandal et al. (\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e), too, showed that ethanol extracts from plant material of \u003cem\u003eBetula papyrifera\u003c/em\u003e inhibited \u003cem\u003eS. aureus\u003c/em\u003e but not \u003cem\u003eE. coli\u003c/em\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e4.2 Implications for Neanderthal healthcare\u003c/h2\u003e\u003cp\u003eThe differences among the six samples collected for our study indicate varying extraction efficiencies, which could be due to factors like the composition of the raw materials, the exact conditions during pyrolysis, or natural variability in the properties of birch bark. Importantly, there is no clear relationship between the extraction method and antibacterial efficacy. Samples produced using the condensation method and the raised structure method result in similar ZOI diameters against gram-positive \u003cem\u003eStaphylococcus aureus\u003c/em\u003e widely known for its role in wound infections. Consequently, the application of birch tar to the skin specifically for the treatment of wounds and skin conditions would have been afforded as early as exploration of its hafting properties has occurred, minimally during MIS 7 from c. 191\u0026ndash;243 ka [5,57], and does not rely on underground pit production. Given the low viscosity of birch tar produced in underground pits, and adhesive properties of birch tar, contamination of the skin during handling is nearly inevitable, regardless of production technique.\u003c/p\u003e\u003cp\u003eIndeed, quantities of birch tar sufficient for skin application are low, with 0.2 g of birch tar covering as much as 100 cm\u003csup\u003e2\u003c/sup\u003e of skin (19; see Fig.\u0026nbsp;4), and are thus easily obtained as a by-product of production for its use in a hafting context, regardless of the production process. In this regard, it might prove useful to consider further uses along with skin application, as previously discussed for ochre (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). For example, studies have suggested the efficacy of birch tar as an insect-repellent [58; Faraone et al., unpublished data], and the Late Pleistocene environments of Europe saw considerable ecological and epidemiological pressure. This is evidenced by, for example, the extensive flying insect assemblages known from Neanderthal sites such as Lichtenberg, or Salzgitter-Lebenstedt (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e) as well as by genetic material relating to immune response introgressed from Neanderthals (\u003cspan additionalcitationids=\"CR63 CR64\" citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eFigure\u0026nbsp;4. A thin layer of viscous birch tar distributed on a white surface.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eApproximately 0.2 g of birch tar are sufficient to cover c. 100 cm\u003csup\u003e2\u003c/sup\u003e of skin surface.\u003c/p\u003e\u003cp\u003eThis study on birch tar\u0026rsquo;s affordances for wound care sits in the context of a surge in interest in Neanderthal life beyond stone tools. Structures of care are increasingly recognized as an essential part of Pleistocene life, and numerous scholars have now published on neglected aspects of Neanderthal care. Spikins et al. (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e) have explored structures of care in the Neanderthal context. Houldcraft and Underdown (\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e) have documented the plethora of pathogens that Neanderthals objected to during the Middle Palaeolithic. Hardy (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e) and Hardy et al. (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e) have documented the manifold plants used potentially medicinally found in Neanderthal contexts. Weyrich et al. (\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e) have documented dental care in a Neanderthal individual from El Sidr\u0026ograve;n. Trinkaus and Villotte (\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e) and Conde-Valverde et al. (\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e) have reported on structures of care related to disability in Neanderthal communities. Today, practice of care is understood to be a key component of Neanderthal life. As such, our study contributes more explicit data on the multimodal affordances of pyrotechnological birch tar production, shedding light on the healthcare of deep time.\u003c/p\u003e\u003c/div\u003e"},{"header":"5 Conclusion","content":"\u003cp\u003eOur experimental findings demonstrate that birch tar possesses selective antibacterial properties, showing consistent inhibitory effects against \u003cem\u003eStaphylococcus aureus\u003c/em\u003e but no detectable activity against \u003cem\u003eEscherichia coli.\u003c/em\u003e Among the six birch tar samples tested, only BT5\u0026mdash;extracted via the raised structure method from \u003cem\u003eBetula pendula\u003c/em\u003e\u0026mdash;showed a comparatively strong response, producing an inhibition zone of 10.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7 mm. Other samples exhibited mild to moderate activity, while one (BT3) showed no effect. This variability underscores how differences in extraction method, bark species, and possibly compound concentration influence the antibacterial efficacy of birch tar. The complete absence of inhibition against \u003cem\u003eE. coli\u003c/em\u003e across all samples aligns with the known structural resistance of Gram-negative bacteria. Further, it supports the tar\u0026rsquo;s specificity toward Gram-positive strains. These results reinforce the hypothesis that birch tar\u0026rsquo;s antibacterial properties may have been exploited intentionally in both deep time and Indigenous contexts for wound care, and bear potential for targeted therapeutic development in the present day.\u003c/p\u003e\u003cp\u003eNonetheless \u0026ndash; our study permits exploring the co-evolutionary relationship between technology and medicine as early as MIS 7, since all three tested production methods showed some level of antibacterial properties, and offers more explicit data to support the medicinal use of antibacterial birch tar in deep time. Whilst drawing from arguments previously proposed for the use of ochre in the Pleistocene, such multimodal uses may indeed be considered for other aspects and localities of Pleistocene lifeways. As such, the use of, for example, biomass adhesive made from \u003cem\u003ePodocarpus\u003c/em\u003e during the Middle Stone Age of southern Africa may also be considered in light of the well-documented antibacterial properties of \u003cem\u003ePodocarpus\u003c/em\u003e plant extract (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan additionalcitationids=\"CR71\" citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e). Yet, further research going beyond the exploratory character of this study is necessary, that further isolates the parameters that affect the antibiotic activity of birch tar produced using different methods.\u003c/p\u003e\u003cp\u003eAs today\u0026rsquo;s world is facing an antibiotic crisis that sees increased antibiotic tolerance of bacterial strains (\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e, \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e), engagement with traditional remedies becomes ever more important. The detection of novel antibacterial remedies (or those that have been neglected in Western medicine) is seen as a key aspect of reconciling with the increasing antibiotic resistance. There is global demand for both broad-spectrum antibiotics that cover both Gram-positive and Gram-negative bacteria strains, as well as more targeted antibacterial remedies that are strain-specific to avoid the administration of broad-spectrum antibiotics, such as the one documented in this study. Along with the affordances of care in deep time explored by our study, this may further lead to the development of therapies specific to \u003cem\u003eS. aureus\u003c/em\u003e.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eAuthor Contribution Statement:\u003c/h2\u003e\u003cp\u003eTS: Conceptualisation, Formal analysis, Investigation, Methodology, Writing - original draft. AO: Data curation, Formal analysis, Investigation, Methodology, Writing - original draft. MS: Investigation, Writing - review \u0026amp; editing. JP: Investigation, Writing - review \u0026amp; editing. MB: Conceptualisation, Funding acquisition, Methodology, Project administration, Supervision, Writing - review \u0026amp; editing.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eTjaark Siemssen is funded by the Boise Trust of the University of Oxford and the German Academic Scholarship Foundation (\u003cem\u003eStudienstiftung\u003c/em\u003e). Aderonke Oludare was funded by Canadian Institutes of Health Research (CIHR) Project Grant 420598 awarded to Matthias Bierenstiel.\u003c/p\u003e\u003ch2\u003eData Availability Statement\u003c/h2\u003e\u003cp\u003eAll relevant data are within the manuscript and its Supporting Information files. Samples of \u003cem\u003eBetula pubescens\u003c/em\u003e and \u003cem\u003eBetula pendula\u003c/em\u003e used in this study can be accessed in the herbarium of the University of Bonn, Germany (herbarium code BONN) via the accession numbers 4155 (\u003cem\u003eB. pendula\u003c/em\u003e) and 4156 (\u003cem\u003eB. pubescens\u003c/em\u003e).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eRoebroeks W, Soressi M. Neandertals revised. Proc Natl Acad Sci USA. 2016;113(23):6372\u0026ndash;9.\u003c/li\u003e\n \u003cli\u003ePawlik AF, Thissen JP. Hafted armatures and multi-component tool design at the Micoquian site of Inden-Altdorf, Germany. Journal of Archaeological Science. 2011;38(7):1699\u0026ndash;708.\u003c/li\u003e\n \u003cli\u003eSchmidt P, Koch TJ, Blessing MA, Karakostis FA, Harvati K, Dresely V, et al. Production method of the K\u0026ouml;nigsaue birch tar documents cumulative culture in Neanderthals. 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Biologia. 2021;76(5):1535\u0026ndash;50.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[{"identity":"10258d64-97d2-4ae7-b67c-9f0d7045884a","identifier":"10.13039/501100000024","name":"Canadian Institutes of Health Research","awardNumber":"420598","order_by":0}],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"University of Oxford","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Early Pyrotechnology, Birch Tar, Ethnopharmacology, Neanderthal Medicine, Transformative Technologies, Experimental Archaeology","lastPublishedDoi":"10.21203/rs.3.rs-8153400/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8153400/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBirch tar is well documented for its use as an adhesive in the Middle Palaeolithic of Europe, but other uses remain poorly explored. Drawing from recent arguments suggesting multimodal uses of products such as ochre and birch tar, this study tests the antibiotic properties of birch tar produced experimentally with methods reconstructed for Middle Palaeolithic birch tar finds from Europe. Made from the bark of \u003cem\u003eBetula pendula\u003c/em\u003e and \u003cem\u003eBetula pubescens\u003c/em\u003e, widely documented for the European Late Pleistocene, we produced birch tar samples using an underground pit method, a condensation method, and a modern tin can method. The birch tar samples were tested for antibiotic properties using the modified Kirby-Bauer disc diffusion antibiotic assay. The results indicate a moderate effect against the Gram-positive \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, a bacterium widely known for its role in wound infections. We further establish that the efficacy of antibiotic properties is not related to the production method, with all methods showing a degree of variation. This supports a coevolutionary relationship between medicinal and technological use and production of birch tar during the Pleistocene. The antibiotic properties documented in this study are consistent with the use of birch tar as a wound dressing and skin ointment in Mi'kmaq communities in Eastern Canada, and the use of birch tar in Saami communities of Lapland. Arguing from an underexplored angle between experimental archaeology and ethnopharmacology, we suggest that similar to the ethnographic examples, a use of birch tar beyond exclusively technological contexts must be considered for the Middle Palaeolithic.\u003c/p\u003e","manuscriptTitle":"Antibacterial properties of experimentally produced birch tar and its medicinal affordances in the Pleistocene","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-01 05:42:38","doi":"10.21203/rs.3.rs-8153400/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7489829c-6222-4acd-b2e3-52051dc532fd","owner":[],"postedDate":"December 1st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-12-01T05:42:38+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-01 05:42:38","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8153400","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8153400","identity":"rs-8153400","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}